Ion generation using modified wetted porous materials

ABSTRACT

The invention generally relates to ion generation using modified wetted porous materials. In certain aspects, the invention generally relates to systems and methods for ion generation using a wetted porous substrate that substantially prevents diffusion of sample into the substrate. In other aspects, the invention generally relate to ion generation using a wetted porous material and a drying agent. In other aspects, the invention generally relates to ion generation using a modified wetted porous substrate in which at least a portion of the porous substrate includes a material that modifies an interaction between a sample and the substrate.

RELATED APPLICATIONS

The present application is a continuation of U.S. nonprovisionalapplication Ser. No. 15/710,072, filed Sep. 20, 2017, which is acontinuation of U.S. nonprovisional application Ser. No. 15/359,031,filed Nov. 22, 2016, which is a is a continuation of U.S. nonprovisionalapplication Ser. No. 14/987,154, filed Jan. 4, 2016, which is a is acontinuation of U.S. nonprovisional application Ser. No. 14/512,579,filed Oct. 13, 2014, which is a continuation of U.S. nonprovisionalapplication Ser. No. 14/119,548, filed Nov. 22, 2013, which is a 35U.S.C. § 371 national phase application of PCT application numberPCT/US12/40521, filed Jun. 1, 2012, which claims the benefit of andpriority to U.S. provisional patent application Ser. No. 61/492,933,filed Jun. 3, 2011, U.S. provisional patent application Ser. No.61/492,937, filed Jun. 3, 2011, and U.S. provisional patent applicationSer. No. 61/492,947, filed Jun. 3, 2011, the content of each of which isincorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under RR031246 andEB009459 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The invention generally relates to systems and methods for massspectrometry analysis of samples.

BACKGROUND

Biofluids (e.g., complex mixtures such as blood, saliva, or urine) areroutinely separated using chromatography before the MS measurement inorder to minimize suppression effects on analyte ionization and topre-concentrate the analytes. Recently, systems and methods have beendeveloped that allow for sample preparation and pre-treatment to becombined with the ionization process (See Ouyang et al., WO2010/127059).

These systems and methods use wetted porous material, named paper sprayionization, for direct, qualitative and quantitative analysis of complexbiofluids (“paper spray”). Analyte transport is achieved by wicking in aporous material with a macroscopically sharp point and a high electricfield is used to perform ionization and chemical analysis of compoundspresent in biological samples. Pneumatic assistance is not required totransport the analyte: a voltage is simply applied to the wet paper,held in front of a mass spectrometer.

SUMMARY

The inventions herein generally relate to various modifications to thewetted porous material used in the paper spray process described above.Such modified substrates lend themselves to more efficient analysis ofcertain samples as well as improved analytical results for thosesamples.

In certain aspects, the invention generally relates to systems andmethods for ion generation using a wetted porous substrate thatsubstantially prevents diffusion of sample into the substrate. Paperspray has been applied for analysis of biofluids, such as dried bloodspots. Generally, the whole blood is deposited onto hydrophilicchromatography paper and dried to form a dried blood spot. The liquidblood is hydrophilic, and when it is dropped onto a hydrophilic poroussample substrate, such as chromatography paper, the fresh blood samplediffuses into the substrate. When the whole blood is dried on the papersubstrate, the microstructure of the paper substrate is modified. Duringthe paper spray, the efficiency of the extraction of chemical from thedried blood spot is limited.

In these embodiments, systems and methods of the invention limit orprevent interaction of the sample with a porous substrate, thus allowingefficient transfer of the sample through the substrate for analysis. Inthese embodiments, the invention provides a mass spectrometry probeincluding at least one porous substrate connected to a high voltagesource, in which the porous substrate includes a material thatsubstantially prevents diffusion of a sample into the substrate. Incertain embodiments, the porous substrate is discrete from a flow ofsolvent. The substrate may have any shape, although shapes having sharppoints are preferred. Exemplary shapes include triangles or cones. Thematerial for the porous substrate will depend on the properties of thesample to be analyzed. For example, when the sample is hydrophilic, thesubstrate is less hydrophilic than the sample. Alternatively, when thesample is hydrophobic, the substrate is less hydrophobic than thesample. An exemplary substrate for analyzing a biological fluid issilanized paper.

In other embodiments, the probe further includes a discrete amount of asolvent, e.g., a droplet or droplets, applied to the porous substrate.The solvent is capable of diffusing into the substrate. The solvent isapplied as a droplet or droplets, and in an amount sufficient to wet theporous substrate. Once applied to the porous substrate, the solvent canassist transport of the sample through the porous material. The solventcan contain an internal standard. The solvent/substrate combination canallow for differential retention of sample components with differentchemical properties. In certain embodiments, the solvent minimizes saltand matrix effects. In other embodiments, the solvent includes chemicalreagents that allow for on-line chemical derivatization of selectedanalytes.

In another embodiment, the invention provides a system for analyzing asample material including at least one porous substrate connected to ahigh voltage source, in which the porous substrate includes a materialthat substantially prevents diffusion of a sample into the substrate,and a mass analyzer. In certain embodiments, the porous substrate isdiscrete from a flow of solvent. The mass analyzer is for a massspectrometer or a handheld mass spectrometer. Exemplary mass analyzersinclude a quadrupole ion trap, a rectalinear ion trap, a cylindrical iontrap, a ion cyclotron resonance trap, an orbitrap, a time of flight, aFourier Transform ion cyclotron resonance, and sectors.

Another embodiment of the invention provides a method for analyzing asample that involves contacting a sample to a porous substrate, in whichthe porous substrate includes a material that substantially preventsdiffusion of the sample into the substrate, applying a solvent that iscapable of diffusing into the substrate to the sample, resulting indiffusion of components of the sample into the substrate, applying ahigh voltage to the substrate to generate ions of the components thatare expelled from the porous substrate, and analyzing the expelled ions.In certain embodiments, the porous substrate is kept separate from aflow of solvent. Exemplary samples include chemical species orbiological species. In certain embodiments, the sample is a biologicalfluid, e.g., complex mixtures such as blood, saliva, or urine.

In certain embodiments, the solvent is capable of mixing with thesample. In other embodiments, the solvent is not capable of mixing withthe sample but is capable of extracting the components from the sample.In certain embodiments, prior to applying the high voltage, the methodfurther involves drying the substrate. In certain embodiments, analyzinginvolves providing a mass analyzer to generate a mass spectrum ofanalytes in the sample.

Another embodiment of the invention provides a method for analyzingblood that involves contacting a blood sample to a porous substrate, inwhich the porous substrate includes a material that substantiallyprevents diffusion of the blood sample into the substrate, applying tothe blood sample a solvent that is capable of diffusing into thesubstrate, resulting in diffusion of components of the blood sample intothe substrate, applying a high voltage to the substrate to generate ionsof the components that are expelled from the porous substrate, andanalyzing the expelled ions. In certain embodiments, the poroussubstrate is kept separate from a flow of solvent. In certainembodiments, the solvent is capable of mixing with the blood sample. Inother embodiments, the solvent is not capable of mixing with the bloodsample but is capable of extracting the components (e.g., proteins) fromthe blood sample. In certain embodiments, prior to applying the highvoltage, the method further involves drying the substrate to produce adried blood spot.

Another embodiment of the invention provides a method for analyzing aprotein from a blood sample that involves contacting a blood samplecomprising at least one protein to a porous substrate, in which theporous substrate includes a material that substantially preventsdiffusion of the blood sample into the substrate, applying to the bloodsample a solvent that is capable of diffusing into the substrate,resulting in diffusion of the protein into the substrate, applying ahigh voltage to the substrate to generate ions of the protein that areexpelled from the porous substrate, and analyzing the expelled ions. Incertain embodiments, the porous substrate is kept separate from a flowof solvent.

Other aspects of the invention generally relate to ion generation usinga wetted porous material and a drying agent. As previously mentioned,paper spray has been applied for analysis of biofluids, such as driedblood spots. One current method for analyzing a dried blood spotinvolves spotting 15 microliters of drug-spiked blood onto Whatman 31ETFcard paper, and allowing it to air dry for at least 2 hours. This dryingtime is important, otherwise the blood can run through the paper duringanalysis, thus reducing the efficiency of the paper spray.

These aspects of the invention combine a drying agent with the poroussubstrate used in paper spray in order to rapidly dry a sample that isapplied to the substrate. Rapid drying decreases waiting time and allowsfor sample analysis within approximately two to five minutes of applyinga sample to the substrate. With respect to analysis of biologicalfluids, probes, systems, and methods of the invention provide apoint-of-care device for rapid and convenient use. In a particularembodiment, probes, systems, and methods of the invention use anhydroussalts to dry and thus restrict flow of liquid blood during paper sprayanalysis.

In certain embodiments, the invention provides a mass spectrometry probeincluding at least one porous substrate connected to a high voltagesource, in which the porous substrate includes a drying agent. Anydrying agent that is compatible with the sample and does not interferewith analysis by mass spectrometry may be used. An exemplary dryingagent is an anhydrous salt. In certain embodiments, the porous substratealso includes an internal standard.

In certain embodiments, the porous substrate is discrete (i.e., separateor disconnected from) from a flow of solvent. Instead, a sample iseither spotted onto the porous substrate or the porous substrate iswetted and used to swab a surface containing the sample. The poroussubstrate with spotted or swabbed sample is then wetted and connected toa high voltage source to produce ions of the sample which aresubsequently analyzed. The sample is transported through the poroussubstrate without the need of a separate solvent flow.

Probes, systems, and methods of the invention combine sample preparationand pre-treatment with the ionization process needed for mass analysisof samples. Probes, systems, and methods of the invention allow forrapid and direct analysis of chemicals in raw biological samples ofcomplex matrices, such as biofluids and tissues, without samplepreparation. In particular embodiments, probes, systems, and methods ofthe invention allow for the analysis of a dried spots of blood or urine.

Exemplary porous materials include paper, e.g., filter paper, or PVDFmembrane. The porous material can be of any shape. In certainembodiments, the porous material is provided as a triangular piece orcone.

In certain embodiments, the probe further includes a discrete amount ofa solvent, e.g., a droplet or droplets, applied to the porous material.The solvent is applied as a droplet or droplets, and in an amountsufficient to wet the porous material. Once applied to the porousmaterial, the solvent can assist transport of the sample through theporous material. The solvent can contain an internal standard. Thesolvent/substrate combination can allow for differential retention ofsample components with different chemical properties. In certainembodiments, the solvent minimizes salt and matrix effects. In otherembodiments, the solvent includes chemical reagents that allow foron-line chemical derivatization of selected analytes.

Another embodiment of the invention provides a system for analyzing asample material including at least one porous substrate connected to ahigh voltage source, in which the porous substrate includes a dryingagent, and a mass analyzer. In certain embodiments, the porous substrateis discrete from a flow of solvent. The mass analyzer is for a massspectrometer or a handheld mass spectrometer. Exemplary mass analyzersinclude a quadrupole ion trap, a rectalinear ion trap, a cylindrical iontrap, a ion cyclotron resonance trap, an orbitrap, a time of flight, aFourier Transform ion cyclotron resonance, and sectors.

Another embodiment of the invention provides a method for analyzing asample that involves contacting a sample to a porous substrate, in whichthe porous substrate includes a drying agent, applying a high voltage tothe porous material to generate ions of an analyte in the sample thatare expelled from the porous material, and analyzing the expelled ions.In certain embodiments, the porous substrate is kept separate from aflow of solvent. In certain embodiments, methods of the inventionfurther involve, prior to applying the high voltage, drying thesubstrate. In certain embodiments, methods of the invention furtherinvolve applying a solvent to the substrate. The solvent is applied as adroplet or droplets, and in an amount sufficient to wet the porousmaterial. Once applied to the porous material, the solvent can assisttransport of the sample through the porous material. In certainembodiments, analyzing involves providing a mass analyzer to generate amass spectrum of analytes in the sample. Exemplary samples includechemical species or biological species. In certain embodiments, thesample is a biological fluid, e.g., complex mixtures such as blood,saliva, or urine.

Another embodiment of the invention involves contacting a blood sampleto a porous substrate, in which the porous substrate includes a dryingagent, drying the blood sample on the substrate, applying a solvent tothe substrate, applying a high voltage to the substrate to generate ionsof the components that are expelled from the porous substrate, andanalyzing the expelled ions. In certain embodiments, applying thesolvent and the high voltage occur simultaneously. In certainembodiments, applying the solvent and the high voltage occursequentially.

Other aspects of the invention generally relate to ion generation usinga modified wetted porous substrate. These aspects of the inventionrecognize that the porous material used in paper spray plays animportant role in determining the resolution of target compounds fromcomplex samples and also in determining the transfer efficiency ofanalyte ions. Accordingly, embodiments of the invention provide a massspectrometry probe that includes at least one porous substrate connectedto a high voltage source, in which at least a portion of the poroussubstrate includes a material that modifies an interaction between asample and the substrate. In certain embodiments, the porous substrateis discrete from a flow of solvent. Exemplary porous substrates includepaper, e.g., filter paper, or PVDF membrane. The porous material can beof any shape. In certain embodiments, the porous material is provided asa triangular piece or cone.

In these embodiments, the material may be any material that modifies theinteraction between the sample and the substrate. In certainembodiments, the material modifies the interaction between the sampleand substrate during sample deposition. In other embodiments, thematerial modifies the interaction between the sample and substrateduring sample elution. The material may coat at least a portion of thesubstrate. Alternatively, the material may impregnate at least a portionof the substrate. In certain embodiments, the material is silica. Inparticular embodiments, the silica coats a surface of the substrate.

In certain embodiments, the probe further includes a discrete amount ofa solvent, e.g., a droplet or droplets, applied to the porous material.The solvent is applied as a droplet or droplets, and in an amountsufficient to wet the porous material. Once applied to the porousmaterial, the solvent can assist transport of the sample through theporous material. The solvent can contain an internal standard. Thesolvent/substrate combination can allow for differential retention ofsample components with different chemical properties. In certainembodiments, the solvent minimizes salt and matrix effects. In otherembodiments, the solvent includes chemical reagents that allow foron-line chemical derivatization of selected analytes.

Another embodiment of the invention provides a system for analyzing asample material including at least one porous substrate connected to ahigh voltage source, in which at least a portion of the porous substrateincludes a material that modifies an interaction between a sample andthe substrate, and a mass analyzer. In certain embodiments, the poroussubstrate is discrete from a flow of solvent. The mass analyzer is for amass spectrometer or a handheld mass spectrometer. Exemplary massanalyzers include a quadrupole ion trap, a rectalinear ion trap, acylindrical ion trap, a ion cyclotron resonance trap, an orbitrap, atime of flight, a Fourier Transform ion cyclotron resonance, andsectors.

Another embodiment of the invention provides a method for analyzing asample that involves contacting a sample to a porous substrate, in whichat least a portion of the porous substrate includes a material thatmodifies an interaction between a sample and the substrate, applying ahigh voltage to the porous material to generate ions of an analyte inthe sample that are expelled from the porous material, and analyzing theexpelled ions. In certain embodiments, the porous substrate is keptseparate from a flow of solvent. Exemplary samples include chemicalspecies or biological species. In certain embodiments, the sample is abiological fluid, e.g., complex mixtures such as blood, saliva, orurine.

Another embodiment of the invention provides a method for analyzingblood that involves contacting a blood sample to a porous substrate, inwhich at least a portion of the porous substrate comprises a silicacoating that modifies an interaction between the blood sample and thesubstrate, applying a high voltage to the porous substrate to generateions of an analyte in the blood sample that are expelled from the poroussubstrate, and analyzing the expelled ions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a drawing of a sample solution being fed to a piece of paperfor electrospray ionization. FIG. 1B is a drawing of a sample solutionpre-spotted onto the paper and a droplet of solvent being subsequentlysupplied to the paper for electrospray ionization.

FIG. 2A is a MS spectrum of heroin (concentration: 1 ppm, volume: 10 μl,solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of the invention.FIG. 2B is a MS/MS spectrum of heroin (concentration: 1 ppb, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 3A is a MS spectrum of caffeine (concentration: 10 ppm, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of theinvention. FIG. 3B is a MS/MS spectrum of caffeine (concentration: 10ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 4A is a MS spectrum of benzoylecgonine (concentration: 10 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 4B is a MS/MS spectrum of benzoylecgonine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 5A is a MS spectrum of serine (concentration: 1 ppm, volume: 10 μl,solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of the invention.FIG. 5B is a MS/MS spectrum of serine (concentration: 100 ppb, volume:10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 6A is a MS spectrum of peptide bradykinin2-9 (concentration: 10ppm, volume: 10 al, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) usingprobes of the invention. FIG. 6B is a MS/MS spectrum of bradykinin2-9(concentration: 1 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 7A is a MS/MS spectrum showing that heroin can be detected fromwhole blood sample by a “spot” method. FIG. 7B shows the MS/MS spectrumof the blood spot without heroin.

FIG. 8A MS/MS spectrum shows heroin can be detected from raw urinesample by a “spot” method. FIG. 8B shows the MS/MS spectrum of the urinespot without heroin.

FIG. 9A is a MS spectrum showing the caffeine detected from a cola drinkwithout sample preparation. FIG. 9B is a MS spectrum showing caffeinedetected from coffee powder. A paper slice was used to collect thecoffee powder from a coffee bag by swabbing the surface.

FIGS. 10A-B shows MS spectra of urine analysis without samplepreparation. FIG. 10A is a MS spectrum showing that caffeine wasdetected in urine from a person who consumed coffee. FIG. 10B is a MSspectrum showing that caffeine was not detected in urine from a personwho had not consumed any coffee.

FIGS. 11A-B are MS spectra showing the difference between peptideanalysis (10 ppm of bradykinin 2-9) on (FIG. 11A) paper triangle and(FIG. 11B) PVDF membrane using the same parameters (˜2 kV, Solvent:MeOH:H₂O=1:1).

FIGS. 12A-D shows direct MS spectra of plant tissues using slicedtissues of four kinds of plants. (FIG. 12A) Onion, (FIG. 12B) Springonion, and two different leaves (FIG. 12C) and (FIG. 12D).

FIGS. 13A-B show MS/MS spectra of Vitamin C. FIG. 13A direct analysis ofonion without sample preparation. FIG. 13B using standard solution.

FIG. 14A is a picture showing dried blood spot analysis on paper; 0.4 μLof whole blood is applied directly to a triangular section ofchromatography paper (typically height 10 mm, base 5 mm). A copper clipholds the paper section in front of the inlet of an LTQ massspectrometer (Thermo Fisher Scientific, San Jose, Calif.) and a DCvoltage (4.5 kV) is applied to the paper wetted with 10 μLmethanol/water (1:1 v/v). FIG. 14B shows the molecular structure ofimatinib (GLEEVEC) and paper spray tandem mass spectrum of 0.4 μL wholeblood containing 4 μg/mL imatinib. Imatinib is identified and quantified(inset) by the MS/MS transition m/z 494→m/z 394 (inset). FIG. 14C showsa quantitative analysis of whole blood spiked with imatinib (62.5-4μg/mL) and its isotopomers imatinib-d8 (1 μg/mL). Inset plot shows lowconcentration range.

FIG. 15 is a paper spray mass spectrum of angiotensin I solution. Theinset shows an expanded view over the mass range 630-700.

FIG. 16 is a mass spectrum showing direct analysis of hormones in animaltissue by probes of the invention.

FIGS. 17A-B are mass spectra showing direct analysis of human prostatetumor tissue and normal tissue.

FIG. 18 is a mass spectrum of whole blood spiked with 10 μg/mL atenolol.The data was obtained by combining systems and methods of the inventionwith a handheld mass spectrometer.

FIGS. 19A-F show mass spectra of cocaine sprayed from six differenttypes of paper (Whatman filter paper with different pore sizes: (FIG.19A) 3 μm, (FIG. 19B) 4-7 μm, (FIG. 19C) 8 μm, and (FIG. 19D) 11 μm,(FIG. 19E) glass fiber paper and (FIG. 19F) chromatography paper). Thespray voltage was 4.5 kV.

FIG. 20A shows a schematic setup for characterizing the spatialdistribution of paper spray. FIG. 20B is a 2D contour plot showing therelative intensity of m/z 304 when the probe is moved in the x-y planewith respect to the inlet of the mass spectrometer. FIG. 20C is a graphshowing signal duration of m/z 304 when loading cocaine solution onpaper with different concentrations or volumes, or sealed by Teflonmembrane.

FIGS. 21A-D are a set of MS spectra of pure chemical solutions and theircorresponding MS/MS spectra. Spectra were obtained for (FIG. 21A)serine, (FIG. 21B) methadone, (FIG. 21C) roxithromycin, and (FIG. 21D)bradykinin 2-9.

FIGS. 22A-G are a set of mass spectra showing analysis of chemicals fromcomplex mixtures and direct analysis from surfaces without samplepreparation. FIGS. 22A-B are mass spectra of COCA-COLA (cola drink),which was directly analyzed on paper in both of (A) positive and (B)negative mode. FIG. 22C is a mass spectrum of caffeine. FIG. 22D is amass spectrum of potassium benzoate. FIG. 22E is a mass spectrum ofacesulfame potassium. FIG. 22F is a mass spectrum of caffeine detectedfrom urine. FIG. 22G is a mass spectrum of heroin detected directly froma desktop surface after swabbing of the surface by probes of theninvention.

FIG. 23A shows images of a probe of the invention used for bloodanalysis. In this embodiment, the porous material is paper. The panel onthe left is prior to spotting with whole blood. The panel in the middleis after spotting with whole blood and allowing the spot to dry. Thepanel on the right is after methanol was added to the paper and allowedto travel through the paper. The panel on the right shows that themethanol interacts with the blood spot, causing analytes to travel tothe tip of the paper for ionization and analysis. FIG. 23B is a massspectrum of Atenolol from whole blood. FIG. 23C is a mass spectrum ofheroin from whole blood.

FIGS. 24A-C show analysis of two dyes, methylene blue (m/z 284) andmethyl violet (m/z 358.5), separated by TLC. Dye mixture solution (0.1μl of a 1 mg/mL solution) was applied onto the chromatography paper (4cm×0.5 cm) and dried before TLC and paper spray MS analysis.

FIGS. 25A-E show different shapes, thicknesses, and angles for probes ofthe invention. FIG. 25A shows sharpness. FIG. 25B shows angle of thetip. FIG. 25C shows thickness of the paper. FIG. 25D shows a device withmultiple spray tips. FIG. 25E shows a DBS card with micro spray tipsfabricated with sharp needles.

FIGS. 26A-B are a set of mass spectra of imatinib from human serum usingdirect spray from a C4 zip-tip of conical shape. Human serum samples(1.5 μL each) containing imatinib were passed through the porous C4extraction material three times and then 3 μL methanol was added ontothe zip-tip with 4 kV positive DC voltage applied to produce the spray.FIG. 26A shows a MS spectrum for 5 μg/mL. FIG. 26B shows a MS/MSspectrum for 5 ng/mL.

FIG. 27A is a picture showing different tip angles for probes of theinvention. From left to right, the angles are 30, 45, 90, 112, 126degree, respectively. FIG. 27B is a graph showing the effect of angle onMS signal intensity. All MS signals were normalized to the MS signalusing the 90 degree tip.

FIG. 28A is a picture of a high-throughput probe device of theinvention. FIG. 28B shows spray from a single tip of the device into aninlet of a mass spectrometer. FIG. 28C is a set of mass spectra showingMS signal intensity in high-throughput mode.

FIG. 29A is a schematic depicting a protocol for direct analysis ofanimal tissue using probes of the invention. FIGS. 29B-D are massspectra showing different chemicals detected in the tissue.

FIG. 30A shows a mass spectral analysis of a dried serum spot on plainpaper. FIG. 30B shows a mass spectrum analysis of a dried serum sport onpaper preloaded with betaine aldehyde (BA) chloride. FIG. 30C shows aMS/MS analysis of reaction product [M+BA]⁺ (m/z 488.6).

FIGS. 31A-B show MS/MS spectra recorded with modified (FIG. 31A) andunmodified (FIG. 31B) paper substrates.

FIG. 32 is a mass spectrum showing that ions can be generated using anegative ion source potential but positively charged ions aremass-analyzed.

FIG. 33A is a schematic showing the design of a sample cartridge withvolume control and overflowing vials. A soluble plug with internalstandard chemical is used to block the bottom of the volume controlvial. FIG. 33B shows a step-by-step process of applying blood samplesonto the cartridge to prepare a dried blood spot on paper from acontrolled volume of blood.

FIGS. 34A-B show mass spectra of agrochemicals that are present on alemon peel purchased from a grocery store and swabbed with paper.

FIG. 35 shows a design of a substrate for paper spray with multiplecorners. The angle of the corner to be used for spray is smaller thanthat of other corners.

FIGS. 36A-B show a spray tip fabricated on a piece of chromatographypaper using SU-8 2010 photoresist. FIG. 36C shows a MS spectrum ofmethanol/water solution containing a mixture of asparagines.

FIGS. 37A-B are schematics showing result of applying solvent too early(<2 hours) to blood spot on paper (FIG. 37A) compared to methods ofpre-loading the paper with a drying agent before spotting the blood,allowing for more rapid analysis (FIG. 37B).

FIG. 38 is a graph showing calibration curves for Tamoxifen by thestandard air-dry method versus quick analysis by pre-spotting MgSO₄.

FIG. 39 is a schematic showing work flow for paper spray analysis ofblood using a drying agent.

FIG. 40 is a schematic showing the mode of operation for point-of-caretherapeutic drug monitoring of whole blood by paper spray massspectrometry utilizing a coagulating agent.

FIG. 41 is a photograph showing paper spray mass spectrometry of 10 μLwhole blood with and without alum, a coagulant which was pre-spotted toclot the blood during analysis.

FIGS. 42A-C show paper spray mass spectrometry of five individualsamples of 50 ng/mL pazopanib (in 10 μL fresh blood) and 5 μL of 500ng/mL ²H₃ ¹³C-pazopanib (pre-spotted). Total mass of pazopanib and ²H₃¹³C-pazopanib deposited onto the paper was 500 pg and 2.5 ng,respectively. (FIG. 42A) SRM ion chronogram of ²H₃ ¹³C-pazopanib (m/z442.1→361.1). (FIG. 42B) SRM ion chronogram of pazopanib (m/z438.1→357.1). (FIG. 42C) Drug-to-internal standard ratio over time.

FIG. 43 shows paper spray mass spectrometry of 1 μg/mL tamoxifen,pazopanib, and irinotecan in 10 μL fresh whole blood with 0.45 mg ofalum was pre-spotted. The spray solvent was 32 μL 50:50methanol/acetonitrile (ACN) and +3.0 kV applied. Abbreviation PC denotesphosphatidylcholine.

FIG. 44A is a photograph showing 10 μL bovine blood on print paper(Xerox 3R2047). FIG. 44B Blood diffuses after 50 μL methanol with 1%acetic acid is applied onto the printing paper.

FIG. 45 is a schematic showing chemical extraction from fresh wholeblood.

FIGS. 46A-C are a set of mass spectra showing detection of proteinhemoglobin from fresh bovine whole blood using different papersubstrates (FIG. 46A) Grade 1 chromatography paper, (FIG. 46B) printerpaper, and (FIG. 46C) silanized paper were used as paper substratesaccordingly.

FIG. 47A is a graph showing quantitation of cotinine, the majormetabolite of nicotine, from fresh bovine whole blood. FIG. 47B is aphotograph of the spray plume using 90% dichloromethane and 10%isopropanol as solvent.

FIG. 48A is a graph showing quantitation of nicotine from fresh bovinewhole blood. FIG. 48B is a photogragh of the spray plume usingacetonitrile as solvent.

FIG. 49 is a graph showing 10 μL whole fresh blood is spotted on to theprint paper.

FIG. 50 shows structures of nicotine, cotinine,trans-3′-hydroxycotinine, and anabasine.

FIGS. 51A-D are a set of graphs showing calibration curves of nicotine(FIG. 51A), cotinine (FIG. 51B), 3HC (FIG. 51C) and anabasine (FIG. 51D)in dried blood spots. The blood samples were spiked with each chemicaland its isotope labeled internal standard (100 ng/mL). Inset plot showslow concentration range. The bars represent the standard deviation ofanalysis for three replicates at different concentrations.

FIG. 52A Chemical extraction during paper spray ionization from freshliquid blood. FIGS. 52B and D: The blood film on the printer paperbefore and after spray. FIG. 52C: Quantitative analysis of fresh liquidblood spiked with nicotine (0.1 ng/mL-100 ng/mL) and its internalstandard nicotine-d3 (100 ng/mL). Inset plot shows low concentrationrange. The bars represent the standard deviation of analysis for threereplicates at different nicotine concentrations.

FIG. 53 shows the direct detection of protein hemoglobin from freshliquid blood on hydrophobic paper substrate. 5 μL blood was applied onthe hydrophobic paper forming a blood droplet. 10 μL methanol wasapplied as spray solvent. The alpha and beta chain and the heme groupfrom hemoglobin could be directly detected.

FIG. 54 shows the calibration curves of cotinine in fresh liquid saliva(1 ng/mL-200 ng/mL) and urine (1 ng/mL-100 ng/mL) using printer paper.The bars represent the standard deviation of analysis for threereplicates at different nicotine concentrations.

FIGS. 55A-I are a set of SEM and photographic images of Grade 4chromatography and silica coated papers without and with dried bloodspots: SEM images of (FIG. 55A) chromatography and (FIG. 55B) silicacoated paper, and (FIG. 55C) close-up image of the selected area in(FIG. 55B) without dried blood spots; top view of (FIG. 55D)chromatography and (FIG. 55E) silica coated paper with dried bloodspots; photograph images of the (FIG. 55F) top and (FIG. 55G) back sidesof chromatography paper, and (FIG. 55H) top and (FIG. 55I) back sides ofsilica coated paper with blood spots.

FIG. 56A Effect of spray solvent on the analysis of verapamil [(M+H)+,m/z 455, product ion, m/z 303] and (FIG. 56B) effect of isopropanolpercentage in dichloromethane on the signal of verapamil with a triplequadrupole. Silica-coated paper substrate used. The peak intensity is anaverage of total ion chronogram values. The concentration of verapamilin the blood sample was 500 ng mL-1.

FIGS. 57A-F are graphs showing a comparison of the elution behavior ofverapamil with silica coated paper and chromatography papers: (i)verapamil in pure water (5 μL, 500 ng mL-1) deposited onto surface of(FIG. 57A) silica coated paper, (FIG. 57B) Grade 4 chromatography paperand (FIG. 57C) ET31 chromatography paper; (ii) verapamil in blood (5 μL,500 ng mL-1) deposited onto surface of (FIG. 57D) silica coated paperand (FIG. 57E) Grade 4 chromatography paper and (FIG. 57F) Grade ET31chromatography paper. Note: the experiments were performed after thepaper had dried. Solvent for silica coated paper was 9:1dichloromethane/isopropanol, chromatography paper 9:1 methanol/water.a.u.: arbitrary units.

FIGS. 58A-C are graphs showing a comparison of the LOQ and lineardynamic range of verapamil with (FIG. 58A) silica coated paper (0.27 mmthick; 9:1 dichloromethane/isopropanol), (FIG. 58B) Grade 4chromatography paper (0.21 mm thick; 9:1 methanol/water), and (FIG. 58C)Grade ET31 chromatography paper (0.50 mm thick; 9:1 methanol/water).Note: 5 μl of blood sample was used, product ion m/z 303 of verapamilwas monitored.

FIGS. 59A-B are graphs showing a linear dynamic range for (FIG. 59A)lidocaine and (FIG. 59A) verapamil, and typical spectra of lidocaine(FIG. 59C) and verapamil (FIG. 59D) with concentrations in blood of 20ng mL-1 and 10 ng mL-1, respectively, obtained with Mini 11.Silica-coated paper (0.27 mm thick) with 9:1dichloromethane/isopropanol.

DETAILED DESCRIPTION

The invention generally relates to ion generation using modified wettedporous materials. In certain aspects, the invention generally relates tosystems and methods for ion generation using a wetted porous substratethat substantially prevents diffusion of sample into the substrate. Inother aspects, the invention generally relate to ion generation using awetted porous material and a drying agent. In other aspects, theinvention generally relates to ion generation using a modified wettedporous substrate in which at least a portion of the porous substrateincludes a material that modifies an interaction between a sample andthe substrate.

A method of generating ions from fluids and solids for mass spectrometryanalysis is described. Porous materials, such as paper (e.g. filterpaper or chromatographic paper) or other similar materials are used tohold and transfer liquids and solids, and ions are generated directlyfrom the edges of the material when a high electric voltage is appliedto the material (FIG. 1). In certain embodiments, the porous material iskept discrete (i.e., separate or disconnected) from a flow of solvent,such as a continuous flow of solvent. Instead, sample is either spottedonto the porous material or swabbed onto it from a surface including thesample. In other embodiments, the paper substrate is directly connectedto a continuous flow of solvent. Further description is provided forexample in Ouyang et al., WO 2010/127059, the content of which isincorporated by reference herein in its entirety.

In certain embodiments, the porous material is any cellulose-basedmaterial. In other embodiments, the porous material is a non-metallicporous material, such as cotton, linen wool, synthetic textiles, orplant tissue. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (5 μm), Grade 595(4-7 μm), and Grade 6 (3 μm), Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultrafiltration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

In certain embodiments, the substrate includes a material thatsubstantially prevents diffusion of a sample into the substrate. Thematerial for the porous substrate will depend on the properties of thesample to be analyzed. For example, when the sample is hydrophilic, thesubstrate is less hydrophilic than the sample. Alternatively, when thesample is hydrophobic, the substrate is less hydrophobic than thesample. Thus the sample remains substantially on top of the substrateuntil an appropriate solvent is applied to the substrate. The solvent iscapable of diffusing into the substrate. The solvent interacts with thesample, causing the sample or components of the sample to diffuse intothe substrate. In certain embodiments, the solvent is capable of mixingwith the sample and both the solvent and the sample diffuse into thesubstrate. In other embodiments, the solvent is not capable of mixingwith the sample but is capable of extracting the components from thesample that diffuse into the substrate along with the solvent.

In other embodiments, the porous material includes a drying agent inorder to rapidly dry a sample that is applied to the substrate. Anydrying agent that is compatible with the sample and does not interferewith analysis by mass spectrometry may be used. An exemplary dryingagent is an anhydrous salt, such as magnesium sulfate, sodium sulfate,sodium carbonate, or calcium chloride. Other exemplary drying agentsinclude blood coagulants, such as alum powder. In certain embodiments,the porous substrate also includes an internal standard. Rapid dryingdecreases waiting time and allows for sample analysis withinapproximately two to five minutes of applying a sample to the substrate.With respect to analysis of biological fluids, such a substrate allowsfor a point-of-care device for rapid and convenient use.

In other embodiments, the substrate includes a material that modifies aninteraction between the sample and the substrate. The material may beany material that modifies the interaction between the sample and thesubstrate. In certain embodiments, the material modifies the interactionbetween the sample and substrate during sample deposition. In otherembodiments, the material modifies the interaction between the sampleand substrate during sample elution. The material may coat at least aportion of the substrate. Alternatively, the material may impregnate atleast a portion of the substrate. In certain embodiments, the materialis silica. In particular embodiments, the silica coats a surface of thesubstrate.

The substrate is then connected to a high voltage source to produce ionsof the sample which are subsequently mass analyzed. The sample istransported through the porous material without the need of a separatesolvent flow. Pneumatic assistance is not required to transport theanalyte; rather, a voltage is simply applied to the porous material thatis held in front of a mass spectrometer.

In certain embodiments, the porous material is integrated with a solidtip having a macroscopic angle that is optimized for spray. In theseembodiments, the porous material is used for filtration,pre-concentration, and wicking of the solvent containing the analytesfor spray at the solid type.

Probes of the invention work well for the generation of micron scaledroplets simply based on using the high electric field generated at anedge of the porous material. In particular embodiments, the porousmaterial is shaped to have a macroscopically sharp point, such as apoint of a triangle, for ion generation. Probes of the invention mayhave different tip widths. In certain embodiments, the probe tip widthis at least about 5 μm or wider, at least about 10 μm or wider, at leastabout 50 μm or wider, at least about 150μm or wider, at least about 250μm or wider, at least about 350 μm or wider, at least about 400 orwider, at least about 450 μm or wider, etc. In particular embodiments,the tip width is at least 350 μm or wider. In other embodiments, theprobe tip width is about 400 m. In other embodiments, probes of theinvention have a three dimensional shape, such as a conical shape.

As mentioned above, no pneumatic assistance is required to transport thedroplets. Ambient ionization of analytes is realized on the basis ofthese charged droplets, offering a simple and convenient approach formass analysis of solution-phase samples.

Sample solution is directly applied on the porous material held in frontof an inlet of a mass spectrometer without any pretreatment. Then theambient ionization is performed by applying a high potential on thewetted porous material. In certain embodiments, the porous material ispaper, which is a type of porous material that contains numerical poresand microchannels for liquid transport. The pores and microchannels alsoallow the paper to act as a filter device, which is beneficial foranalyzing physically dirty or contaminated samples.

In other embodiments, the porous material is treated to producemicrochannels in the porous material or to enhance the properties of thematerial for use as a probe of the invention. For example, paper mayundergo a patterned silanization process to produce microchannels orstructures on the paper. Such processes involve, for example, exposingthe surface of the paper totridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to result insilanization of the paper. In other embodiments, a soft lithographyprocess is used to produce microchannels in the porous material or toenhance the properties of the material for use as a probe of theinvention. In other embodiments, hydrophobic trapping regions arecreated in the paper to pre-concentrate less hydrophilic compounds.

Hydrophobic regions may be patterned onto paper by usingphotolithography, printing methods or plasma treatment to definehydrophilic channels with lateral features of 200˜1000 m. See Martinezet al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al.(Proc. Natl Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal.Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80,3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li et al.(Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. Liquid samples loadedonto such a paper-based device can travel along the hydrophilic channelsdriven by capillary action.

Another application of the modified surface is to separate orconcentrate compounds according to their different affinities with thesurface and with the solution. Some compounds are preferably absorbed onthe surface while other chemicals in the matrix prefer to stay withinthe aqueous phase. Through washing, sample matrix can be removed whilecompounds of interest remain on the surface. The compounds of interestcan be removed from the surface at a later point in time by otherhigh-affinity solvents. Repeating the process helps desalt and alsoconcentrate the original sample.

In certain embodiments, methods and systems of the invention use aporous material, e.g., paper, to hold and transport analytes for massspectral analysis. Analytes in samples are pre-concentrated, enrichedand purified in the porous material in an integrated fashion forgeneration of ions with application of a high voltage to the porousmaterial. In certain embodiments, a discrete amount of transportsolution (e.g., a droplet or a few droplets) is applied to assistmovement of the analytes through the porous material. In certainembodiments, the analyte is already in a solution that is applied to theporous material. In such embodiments, no additional solvent need beadded to the porous material. In other embodiments, the analyte is in apowdered sample that can be easily collected by swabbing a surface.Systems and methods of the invention allow for analysis of plant oranimal tissues, or tissues in living organisms.

Methods and systems of the invention can be used for analysis of a widevariety of small molecules, including epinephrine, serine, atrazine,methadone, roxithromycin, cocaine and angiotensin I. All display highquality mass and MS/MS product ion spectra (see Examples below) from avariety of porous surfaces. Methods and systems of the invention allowfor use of small volumes of solution, typically a few μL, with analyteconcentrations on the order of 0.1 to 10 μg/mL (total amount analyte 50pg to 5 ng) and give signals that last from one to several minutes.

Methods and systems of the invention can be used also for analysis of awide variety of biomolecules, including proteins and peptides. Methodsof the invention can also be used to analyze oligonucleotides from gels.After electrophoretic separation of oligonucleotides in the gel, theband or bands of interest are blotted with porous material using methodsknown in the art. The blotting results in transfer of at least some ofthe oligonucleotides in the band in the gel to the porous material. Theporous material is then connected to a high voltage source and theoligonucleotides are ionized and sprayed into a mass spectrometer formass spectral analysis.

Methods and systems of the invention can be used for analysis of complexmixtures, such as whole blood or urine. The typical procedure for theanalysis of pharmaceuticals or other compounds in blood is a multistepprocess designed to remove as many interferences as possible prior toanalysis. First, the blood cells are separated from the liquid portionof blood via centrifugation at approximately 1000×g for 15 minutes(Mustard, J. F.; Kinlough-Rathbone, R. L.; Packham, M. A. Methods inEnzymology; Academic Press, 1989). Next, the internal standard is spikedinto the resulting plasma and a liquid-liquid or solid-phase extractionis performed with the purpose of removing as many matrix chemicals aspossible while recovering nearly all of the analyte (Buhrman, D. L.;Price, P. I.; Rudewicz, P. J. Journal of the American Society for MassSpectrometry 1996, 7, 1099-1105). The extracted phase is typically driedby evaporating the solvent and then resuspended in the a solvent used asthe high performance liquid chromatography (HPLC) mobile phase(Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Ithaca, N.Y.,Jul. 23-25 1997; 882-889). Finally, the sample is separated in thecourse of an HPLC run for approximately 5-10 minutes, and the eluent isanalyzed by electrospray ionization-tandem mass spectrometry(Hopfgartner, G.; Bourgogne, E. Mass Spectrometry Reviews 2003, 22,195-214).

Methods and systems of the invention avoid the above sample work-upsteps. Methods and systems of the invention analyze a dried blood spotsin a similar fashion, with a slight modification to the extractionprocedure. First, a specialized device is used to punch out identicallysized discs from each dried blood spot. The material on these discs isthen extracted in an organic solvent containing the internal standard(Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clinical Chemistry 2003, 49,1797-1817). The extracted sample is dried on the paper substrate, andthe analysis proceeds as described herein.

Examples below show that methods and systems of the invention candirectly detect individual components of complex mixtures, such ascaffeine in urine, 50 pg of cocaine on a human finger, 100 pg of heroinon a desktop surface, and hormones and phospholipids in intact adrenaltissue, without the need for sample preparation prior to analysis (SeeExamples below). Methods and systems of the invention allow for simpleimaging experiments to be performed by examining, in rapid succession,needle biopsy tissue sections transferred directly to paper.

Analytes from a solution are applied to the porous material forexamination and the solvent component of the solution can serve as theelectrospray solvent. In certain embodiments, analytes (e.g., solid orsolution) are pre-spotted onto the porous material, e.g., paper, and asolvent is applied to the material to dissolve and transport the analyteinto a spray for mass spectral analysis.

In certain embodiments, a solvent is applied to the porous material toassist in separation/extraction and ionization. Any solvents may be usedthat are compatible with mass spectrometry analysis. In particularembodiments, favorable solvents will be those that are also used forelectrospray ionization. Exemplary solvents include combinations ofwater, methanol, acetonitrile, and THE The organic content (proportionof methanol, acetonitrile, etc. to water), the pH, and volatile salt(e.g. ammonium acetate) may be varied depending on the sample to beanalyzed. For example, basic molecules like the drug imatinib areextracted and ionized more efficiently at a lower pH. Molecules withoutan ionizable group but with a number of carbonyl groups, like sirolimus,ionize better with an ammonium salt in the solvent due to adductformation.

In certain embodiments, a multi-dimensional approach is undertaken. Forexample, the sample is separated along one dimension, followed byionization in another dimension. In these embodiments, separation andionization can be individually optimized, and different solvents can beused for each phase.

In other embodiments, transporting the analytes on the paper isaccomplished by a solvent in combination with an electric field. When ahigh electric potential is applied, the direction of the movement of theanalytes on paper is found to be related to the polarity of theircharged forms in solution. Pre-concentration of the analyte before thespray can also be achieved on paper by placing an electrode at a pointon the wetted paper. By placing a ground electrode near the paper tip, astrong electric field is produced through the wetted porous materialwhen a DC voltage is applied, and charged analytes are driven forwardunder this electric field. Particular analytes may also be concentratedat certain parts of the paper before the spray is initiated.

In certain embodiments, chemicals are applied to the porous material tomodify the chemical properties of the porous material. For example,chemicals can be applied that allow differential retention of samplecomponents with different chemical properties. Additionally, chemicalscan be applied that minimize salt and matrix effects. In otherembodiments, acidic or basic compounds are added to the porous materialto adjust the pH of the sample upon spotting. Adjusting the pH may beparticularly useful for improved analysis of biological fluids, such asblood. Additionally, chemicals can be applied that allow for on-linechemical derivatization of selected analytes, for example to convert anon-polar compound to a salt for efficient electrospray ionization.

In certain embodiments, the chemical applied to modify the porousmaterial is an internal standard. The internal standard can beincorporated into the material and released at known rates duringsolvent flow in order to provide an internal standard for quantitativeanalysis. In other embodiments, the porous material is modified with achemical that allows for pre-separation and pre-concentration ofanalytes of interest prior to mass spectrum analysis.

The spray droplets can be visualized under strong illumination in thepositive ion mode and are comparable in size to the droplets emittedfrom a nano-electrospray ion sources (nESI). In the negative ion mode,electrons are emitted and can be captured using vapor phase electroncapture agents like benzoquinone. Without being limited by anyparticular theory or mechanism of action, it is believed that the highelectric field at a tip of the porous material, not the fields in theindividual fluid channels, is responsible for ionization.

The methodology described here has desirable features for clinicalapplications, including neotal screening, therapeutic drug monitoringand tissue biopsy analysis. The procedures are simple and rapid. Theporous material serves a secondary role as a filter, e.g., retainingblood cells during analysis of whole blood. Significantly, samples canbe stored on the porous material and then analyzed directly from thestored porous material at a later date without the need transfer fromthe porous material before analysis. Systems of the invention allow forlaboratory experiments to be performed in an open laboratoryenvironment.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

The following examples are intended to further illustrate certainembodiments of the invention, and are not to be construed to limit thescope of the invention. Examples herein show that mass spectrometryprobes of the invention can ionize chemical and biological samples,allowing for subsequent mass analysis and detection. An exemplary probewas constructed as a paper triangle, which was used to generate micronscale droplets by applying a high potential on the paper. The analyteswere ionized from these electrically charged droplets and transportedinto a conventional mass spectrometer.

Examples below show that a wide range of samples could be directlyanalyzed in the ambient environment by probes of the invention in bothof pure state and complex mixtures. The results showed that paper-basedspray has the following benefits: it operated without sheath gas, i.e.,few accessories were required for in situ analysis; biological samples(dried blood, urine) could be stored on the precut filter papers formonths before analysis; filter paper minimized matrix effects seen withelectrospray or nano electrospray in many samples (blood cells, salt andproteins) and enhanced the MS signal of chemicals in complex samples;powdered samples were easily collected by swabbing surfaces using paperpieces and then directly analyzed; the paper could be pretreated tocontain internal standards that were released at known rates duringsolvent flow in quantitative analysis; and the paper could be pretreatedto contain matrix suppression or absorption sites or to perform ionexchange or to allow on-line chemical derivatization of selectedanalytes.

Detection of most analytes was achieved as low as ppb levels (whenexamined as solutions) or in the low ng to pg range (when solids wereexamined) and the detection time was less than one minute. CertainExamples below provide a protocol for analyzing a dried blood spot,which can also be used for in situ analysis of whole blood samples. Thedried blood spot method is also demonstrated to be compatible with thestorage and transport of blood sample for blood screening and otherclinical tests.

Devices of the invention integrated the capabilities of sampling,pre-separation, pre-concentration and ionization. Methods and systems ofthe invention simplify the problem of sample introduction in massanalyzers.

Example 1: Construction of an MS Probe

Filter paper was cut into triangular pieces with dimensions of 10 mmlong and 5 mm wide and used as a sprayer (FIG. 1). A copper clip wasattached to the paper, and the paper was oriented to face an inlet of amass spectrometer (FIG. 1). The copper clip was mounted on a 3D movingstage to accurately adjust its position. A high voltage was applied tothe copper clip and controlled by a mass spectrometer to generateanalyte ions for mass detection.

Samples were directly applied to the paper surface that served as asample purification and pre-concentration device. Filter paper allowedliquid samples to move through the hydrophilic network driven bycapillary action and electric effects and to transport them to the tipof the paper. Separation could take place during this transport process.Sample solution was sprayed from the tip and resulted in ionization andMS detection when a high voltage (˜4.5 kV) was applied to the papersurface.

All experiments were carried out with a Finnigan LTQ mass spectrometer(Thermo Electron, San Jose, Calif.). The typical temperature of thecapillary inlet was set at 150° C. while 30° C. for heroin detection.The lens voltage was set at 65 V for sample analysis and 240 V forsurvival yield experiment. Tandem mass spectra were collected usingcollision-induced dissociation (CID) to identify analytes in testedsamples, especially for complex mixtures and blood samples.

Example 2: Spray Generation

Spray was produced by applying a high potential on the wetted papertriangle. One paper triangle was placed in front of the inlet of LTQwith its sharp tip facing to the inlet, separated by 3 mm or more.Typically, 10 uL sample solution was applied to wet the paper triangle.The solution can wet or saturate the paper or form a thin layer ofliquid film on the surface of the paper. A high potential (3-5 kV) wasapplied between the paper triangle and mass inlet to generate anelectric field, which induced a charge accumulation on the liquid at thetip of paper triangle. The increasing coulombic force breaks the liquidto form charged droplets and then the solvent evaporated during theflight of droplets from the paper tip to the mass analyzer. Paper sprayrequired no sheath gas, heating or any other assistance to remove thesolvent.

When liquid accumulated on the paper triangle, a Taylor cone wasobserved at the tip when examined with a microscope. The droplets formedwere clearly visible under strong illumination. The Taylor cone andvisible spray disappeared after a short time of evaporation and spray.However, the mass signal lasted for a much longer period (severalminutes). This revealed that the paper triangle could work in two modesfor mass analysis. In a first mode, the liquid was transported insidethe paper at a rate faster than the liquid could be consumed as spray atthe paper tip, resulting in a large cone being formed at the paper tipand droplets being generated. In a second mode, the liquid transportinside the paper was not able to move at a rate fast enough to keep upwith the spray consumption, and droplets were not visible. However, itwas observed that ionization of analytes did take place. The first modeprovided ESI like mass spectra and the second mode provided spectra withsome of the features APCI spectra. In the latter case, the papertriangle played a role analogous to a conductive needle to generate ahigh electric field to ionize the molecules in the atmosphere. It wasobserved that the mass signal in the first mode was stronger than themass signal in the second mode by approximately two orders of magnitudeunder the conditions and for the samples tested.

Example 3: Probe Considerations

a. Probe Materials

A number of porous materials were tested to generate charged dropletsfor mass spectrometry. The materials were shaped into triangles havingsharp tips and sample solution was then applied to the constructedprobes. Data herein show that any hydrophilic and porous substrate couldbe used successfully, including cotton swab, textile, plant tissues aswell as different papers. The porous network or microchannels of thesematerials offered enough space to hold liquid and the hydrophilicenvironment made it possible for liquid transport by capillary action.Hydrophobic and porous substrates could also be used successfully withproperly selected hydrophobic solvents.

For further investigation, six kinds of commercialized papers wereselected and qualitatively tested to evaluate their capabilities inanalyte detection. Filter papers and chromatography paper were made fromcellulose, while glass microfiber filter paper was made from glassmicrofiber. FIG. 19 shows the mass spectra of cocaine detection on thosepapers. The spectrum of glass fiber paper (FIG. 19 panel E) was uniquebecause the intensity of background was two orders of magnitude lowerthan other papers and the cocaine peak (m/z, 304) could not beidentified.

It was hypothesized that the glass fiber paper was working on mode IIand prohibiting efficient droplet generation, due to the relative largethickness (˜2 mm). This hypothesis was proved by using a thin layerpeeled from glass fiber paper for cocaine detection. In that case, theintensity of the background increased and a cocaine peak was observed.All filter papers worked well for cocaine detection, (FIG. 19 panelsA-D). Chromatography paper showed the cleanest spectrum and relativehigh intensity of cocaine (FIG. 19 panel F).

b. Probe Shape and Tip Angle

Many different probe shapes were investigated with respect to generatingdroplets. A preferred shape of the porous material included at least onetip. It was observed that the tip allowed ready formation of a Taylorcone. A probe shape of a triangle was used most often. As shown in FIG.25 panels (A-C), the sharpness of the tip, the angle of the tip (FIG. 27panels A and B), and the thickness of the paper substrate could effectthe spray characteristics. The device of a tube shape with multiple tips(FIG. 25 panel D) is expected to act as a multiple-tip sprayer, whichshould have improved spray efficiency. An array of micro sprayers canalso be fabricated on a DBS card using sharp needles to puncture thesurface (FIG. 25 panel E).

Example 4: Configuration of Probe with Inlet of a Mass Spectrometer

A paper triangle was mounted on a 2D moving stage to determine how themass signal was affected by the relative positions of the paper triangleand the mass spectrometer inlet. The paper triangle was moved 8 cm inthe y-direction in a continuous manner and 3 cm in the x-direction witha 2 mm increment for each step (FIG. 20 panel A). Cocaine solution (1ug/mL, methanol/water, 1:1 v/v) was continuously fed onto the papersurface. The mass spectrum was continuously recorded during the entirescan. A contour plot of the peak intensity of protonated cocaine (m/z,304) was created from the normalized data extracted from the massspectrum (FIG. 20 panel B). The contour plot shows that it was notnecessary for the paper triangle to be placed directly in-line with theinlet of the mass spectrometer to generate droplets.

Spray duration was also tested (FIG. 20 Panel C). Paper triangles (size10 mm, 5 mm) were prepared. First, 10 uL solutions were applied on thepaper triangles with different concentration of 0.1, 1 and 10 ug/mL. Thespray time for each paper was just slightly varied by the difference ofconcentration. After that, 1 ug/mL cocaine solutions were applied on thepaper triangles with different volumes of 5 uL, 10 uL and 15 uL. Thespray times showed a linear response followed by the increasing samplevolumes.

In another test, the paper was sealed with a PTFE membrane to preventevaporation of solution, which prolonged the spray time by about threetimes. These results indicate that paper spray offers long enough timeof spray for data acquisition even using 5 uL solution, and theintensity of signal is stable during the entire spray period.

Example 5: Separation and Detection

Probes of the invention include a porous material, such as paper, thatcan function to both separate chemicals in biological fluids before insitu ionization by mass spectrometry. In this Example, the porousmaterial for the probe was chromatography paper. As shown in FIG. 24, amixture of two dyes was applied to the paper as a single spot. The dyeswere first separated on the paper by TLC (thin layer chromatograph) andthe separated dyes were examined using MS analysis by methods of theinvention with the paper pieces cut from the paper media (FIG. 24). Datashow the separate dyes were detected by MS analysis (FIG. 24).

The chromatography paper thus allowed for sample collection, analyteseparation and analyte ionization. This represents a significantsimplification of coupling chromatography with MS analysis.Chromatography paper is a good material for probes of the inventionbecause such material has the advantage that solvent movement is drivenby capillary action and there is no need for a syringe pump. Anotheradvantage is that clogging, a serious problem for conventionalnanoelectrospray sources, is unlikely due to its multi-porouscharacteristics. Therefore, chromatography paper, a multi-porousmaterial, can be used as a microporous electrospray ionization source.

Example 6: Pure Compounds: Organic Drugs, Amino Acids, and Peptides

As already described, probes and methods of the invention offer a simpleand convenient ionization method for mass spectrometry. Paper triangleswere spotted with different compounds and connected to a high voltagesource to produce ions. All experiments were carried out with a FinniganLTQ mass spectrometer (Thermo Electron, San Jose, Calif.). Data hereinshow that a variety of chemicals could be ionized in solution phase,including amino acid, therapeutic drugs, illegal drugs and peptides.

FIG. 2 panel (A) shows an MS spectrum of heroin (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 2 panel (B) shows MS/MS spectrum of heroin(concentration: 1 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 3 panel (A) shows MS spectrum of caffeine (concentration: 10 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 3 panel (B) shows MS/MS spectrum of caffeine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)). Peak 167 also exists in the blank spectrum with solvent andwithout caffeine.

FIG. 4 panel (A) shows MS spectrum of benzoylecgonine (concentration: 10ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) usingprobes of the invention. FIG. 4 panel (B) shows MS/MS spectrum ofbenzoylecgonine (concentration: 10 ppb, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 5 panel (A) shows MS spectrum of serine (concentration: 1 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 5 panel (B) shows MS/MS spectrum of serine(concentration: 100 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)). Peak 74 and 83 also exist in the blank spectrum with solventand without serine. FIG. 21 panel (A) shows MS spectrum of serine (m/z,106) using probes of the invention. Panel (A) also shows MS/MS spectrumof serine (m/z, 106).

FIG. 21 Panel (B) shows MS spectrum of methadone (m/z, 310) using probesof the invention. Panel (B) also shows MS/MS spectrum of methadone (m/z,310). Panel (C) shows MS spectrum of roxithromycin (m/z, 837) usingprobes of the invention. Panel (B) also shows MS/MS spectrum ofroxithromycin (m/z, 837).

FIG. 6 panel (A) shows MS spectrum of peptide bradykinin2-9(concentration: 10 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)) using probes of the invention. FIG. 6 panel (B) shows MS/MSspectrum of bradykinin2-9 (concentration: 1 ppm, volume: 10 μl, solvent:MeOH/H₂O/HOAc (50:49:1, v/v/v)). The hump in the spectrum is assumed tobe caused by polymers, such as polyethylene glycol (PEG), which arefrequently added to materials in industry. FIG. 21 panel (D) shows MSspectrum of bradykinin 2-9 (m/z, 453) using probes of the invention.Panel (D) also shows MS/MS spectrum of bradykinin 2-9 (m/z, 453). PanelD further shows adduct ions [M+H] (m/z, 904), [M+2H]²⁺ (m/z, 453),[M+H+Na]²⁺ (m/z, 464) and [M+2Na]²⁺ (m/z, 475). The m/z 453 peak wasdouble charged adduct ion confirmed by the MS/MS spectrum.

FIG. 11 is an MS spectra showing the difference between peptide analysis(10 ppm of bradykinin 2-9) on (A) paper slice and (B) PVDF membraneusing the same parameters (˜2 kV, Solvent: MeOH:H₂O=1:1).

Data herein show that probes of the invention work well over themass/charge range from 50 to over 1000 for detection of pure compounds.Data further shows that detection was achieved down to as low as 1 ng/mLfor most chemicals, including illegal drugs, such as heroin, cocaine andmethadone.

Example 7: Complex Mixtures

Complex mixtures such as urine, blood, and cola drink were examinedusing methods, devices, and systems of the invention. All experimentswere carried out with a Finnigan LTQ mass spectrometer (Thermo Electron,San Jose, Calif.).

FIG. 7 panel (A) shows an MS/MS spectrum that shows that heroin wasdetected from whole blood sample by a “spot” method. 0.4 μl of wholeblood sample containing 200 ppb heroin was applied on the center of thetriangle paper to form a 1 mm² blood spot. After the spot was dry, 10 μlof solvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear endof the triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “blood spot” method mentionedabove, the whole blood was added on the paper for electrospray directly.MS/MS spectrum showed that heroin was not detected from 10 μl of wholeblood sample, even when the concentration was as high as 20 ppm (FIG. 7panel B).

FIG. 8 panel (A) shows an MS/MS spectrum that shows that heroin can bedetected from raw urine sample by a “spot” method. 0.4 μl of raw urinesample containing 100 ppb heroin was applied on the center of thetriangle paper to form a 1 mm² urine spot. After the spot was dry, 10 μlof solvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear endof the triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “spot” method mentioned above, theraw urine was added on the paper for electrospray directly. MS/MSspectrum showed heroin was not detected from 10 μl of raw urine samplewhen concentration was 100 ppb (FIG. 8 panel B).

FIG. 9 panel (A) is an MS spectrum showing that caffeine was detectedfrom a cola drink without sample preparation. FIG. 9 panel (B) is an MSspectrum showing that caffeine was detected from coffee powder. A papertriangle was used to collect the coffee powder from a coffee bag byswabbing the surface.

FIG. 22 panels (A-B) show the spectra of COCA-COLA (cola drink),analyzed in positive mode and negative mode, respectively. The peak ofprotonated caffeine, m/z 195, identified in MS/MS spectrum, wasdominated in the mass spectrum in positive mode due to the highconcentration of caffeine (100 ug/mL) in this drink (Panel C). Two highconcentrated compounds, potassium benzoate and acesulfame potassium wereidentified in the MS/MS spectrum in negative mode (Panels D-E).

FIG. 22 panel F shows spectra of caffeine in urine from a person who haddrunk COCA-COLA (cola drink) two hours before the urine collection.Urine typically contains urea in very high concentration, which is alsoeasily ionized. Therefore, protonated urea [m/z, 61] and urea dimmer[m/z, 121] dominated the MS spectrum. However, the protonated caffeinewas identified in the MS/MS spectrum, which showed good signal to noiseratio in the urine sample.

FIG. 10 shows MS spectra of urine taken for analysis without samplepreparation. FIG. 10 panel (A) is a mass spectra of caffeine that wasdetected in urine from a person who had consumed coffee. FIG. 10 panel(B) is a mass spectra showing that caffeine was not detected in urinefrom a person who had not consumed any coffee.

FIG. 22 panel G shows the MS spectrum of heroin (m/z, 370) collected asa swabbed sample. A 5 uL solution containing 50 ng heroin was spotted ona 1 cm² area of a desktop. The paper triangle was wetted and used toswab the surface of the desktop. The paper triangle was then connectedto the high voltage source for mass detection. This data shows thatprobes of the invention can have dual roles of ionization source as wellas a sampling device for mass detection. Trace sample on solid surfacecould be simply collected by swabbing the surface using probes of theinvention. Dust and other interferences were also collected on the papertriangle, but the heroin could be directly detected from this complexmatrix.

Example 8: Plant Tissue Direct Analysis by ESI without Extraction

FIG. 12 shows direct MS spectra of plant tissues using sliced tissues offour kinds of plants. (A) Onion, (B) Spring onion, and two differentleaves (C) and (D).

FIG. 13 shows an MS/MS spectra of Vitamin C analysis (A) direct analysisof onion without sample preparation, (B) using standard solution.

Example 9: Whole Blood and Other Biofluids

Body fluids, such as plasma, lymph, tears, saliva, and urine, arecomplex mixtures containing molecules with a wide range of molecularweights, polarities, chemical properties, and concentrations. Monitoringparticular chemical components of body fluids is important in a numberof different areas, including clinical diagnosis, drug development,forensic toxicology, drugs of abuse detection, and therapeutic drugmonitoring. Tests of blood, including the derived fluids plasma andserum, as well as on urine are particularly important in clinicalmonitoring.

A wide variety of chemicals from blood are routinely monitored in aclinical setting. Common examples include a basic metabolic panelmeasuring electrolytes like sodium and potassium along with urea,glucose, and creatine and a lipid panel for identifying individuals atrisk for cardiovascular disease that includes measurements of totalcholesterol, high density lipoprotein (HDL), low density lipoprotein(LDL), and triglycerides. Most laboratory tests for chemicals in bloodare actually carried out on serum, which is the liquid component ofblood separated from blood cells using centrifugation. This step isnecessary because many medical diagnostic tests rely on colorimetricassays and therefore require optically clear fluids. Aftercentrifugation, detection of the molecule of interest is carried in anumber of ways, most commonly by an immunoassay, such as anenzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), oran enzyme assay in which the oxidation of the molecule of interest by aselective enzyme is coupled to a reaction with a color change, such asthe tests for cholesterol (oxidation by cholesterol oxidase) or glucose(oxidation by glucose oxidase).

There is considerable interest in the pharmaceutical sciences in thestorage and transportation of samples of whole blood as dried bloodspots on paper (N. Spooner et al. Anal Chem., 2009, 81, 1557). Mosttests for chemicals found in blood are carried out on a liquid sample,typically serum or plasma isolated from the liquid whole blood. Therequired storage, transportation, and handling of liquid blood or bloodcomponents present some challenges. While blood in liquid form isessential for some tests, others can be performed on blood or other bodyfluids that have been spotted onto a surface (typically paper) andallowed to dry.

Probes and methods of the invention can analyze whole blood without theneed for any sample preparation. The sample was prepared as follows. 0.4uL blood was directly applied on the center of paper triangle and leftto dry for about 1 min. to form a dried blood spot (FIG. 23 panel A). 10uL methanol/water (1:1, v/v) was applied near the rear end of the papertriangle. Driven by capillary action, the solution traveled across thepaper wetting it throughout its depth. As the solution interacted withthe dried blood spot, the analytes from the blood entered the solutionand were transported to the tip of the probe for ionization (FIG. 23panel A). The process of blood sample analysis was accomplished in about2 min.

Different drugs were spiked into whole blood and the blood was appliedto probes of the invention as described above. Detection of differentdrugs is described below.

Imatinib (GLEEVEC), a 2-phenylaminopyrimidine derivative, approved bythe FDA for treatment of chronic myelogenous leukemia, is efficaciousover a rather narrow range of concentrations. Whole human blood, spikedwith imatinib at concentrations including the therapeutic range, wasdeposited on a small paper triangle for analysis (FIG. 14, panel A). Thetandem mass spectrum (MS/MS, FIG. 14 panel B) of protonated imatinib,m/z 494, showed a single characteristic fragment ion. Quantitation ofimatinib in whole blood was achieved using this signal and that for aknown concentration of imatinib-d8 added as internal standard. Therelative response was linear across a wide range of concentrations,including the entire therapeutic range (FIG. 14 panel C).

Atenolol, a β-blocker drug used in cardiovascular diseases, was testedusing the dried blood spot method to evaluate paper spray for wholeblood analysis. Atenolol was directly spiked into whole blood at desiredconcentrations and the blood sample was used as described above forpaper spray. The protonated atenolol of 400 pg (1 ug/mL atenolol in 0.4uL whole blood) in dried blood spot was shown in mass spectra, and theMS/MS spectra indicated that even 20 pg of atenolol (50 ug/mL atenololin 0.4 uL whole blood) could be identified in the dried blood spot (FIG.23 panel B).

FIG. 23 panel (C) is a mass spectra of heroin in whole blood. Dataherein show that 200 pg heroin in dried blood spot could be detectedusing tandem mass.

It was also observed that the paper medium served a secondary role as afilter, retaining blood cells. Significantly, samples were analyzeddirectly on the storage medium rather than requiring transfer from thepaper before analysis. All experiments were done in the open labenvironment. Two additional features indicated that the methodology hadthe potential to contribute to increasing the use of mass spectrometryin primary care facilities: blood samples for analysis were drawn bymeans of a pinprick rather than a canula; and the experiment was readilyperformed using a handheld mass spectrometer (FIG. 18 and Example 10below).

Example 10: Handheld Mass Spectrometer

Systems and methods of the invention were compatible with a handheldmass spectrometer. Paper spray was performed using a handheld massspectrometer (Mini 10, custom made at Purdue University). Analysis ofwhole blood spiked with 10 μg/mL atenolol. Methanol/water (1:1; 10 μL)was applied to the paper after the blood (0.4 uL) had dried (˜1 min) togenerate spray for mass detection (FIG. 18). The inset shows thatatenolol could readily be identified in whole blood using tandem massspectrum even when the atenolol amount is as low as 4 ng.

Example 11: Angiotensin I

FIG. 15 is a paper spray mass spectrum of angiotensin I solution(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO: 1), 10 μL, 8 μg/mLin methanol/water, 1:1, v/v) on chromatography paper (spray voltage, 4.5kV). The inset shows an expanded view over the mass range 630-700. Theprotonated ([M+2H]²⁺) and sodium-adduct ions ([M+H+Na]²⁺, [M+2Na]²⁺) arethe major ionic species.

Example 12: Agrochemicals on Fruit

Sample collection by paper wiping followed by analysis using probes ofthe invention was used for fast analysis of agrochemicals on fruit.Chromatography paper (3×3 cm) wetted with methanol was used to wipe a 10cm² area on the peel of a lemon purchased from a grocery store. Afterthe methanol had dried, a triangle was cut from the center of the paperand used for paper spray by applying 10 μL methanol/water solution. Thespectra recorded (FIG. 34 panels A-B) show that a fungicide originallyon the lemon peel, thiabendazole (m/z 202 for protonated molecular ionand m/z 224 for sodium adduct ion), had been collected onto the paperand could be identified easily with MS and confirmed using MS/MSanalysis. Another fungicide imazalil (m/z 297) was also observed to bepresent.

Example 13: Tumor Sample

Systems and methods of the invention were used to analyze human prostatetumor tissue and normal tissue. Tumor and adjacent normal tissuesections were 15 μm thick and fixed onto a glass slide for an imagingstudy using desorption electrospray ionization (DESI). A metal needlewas used to remove a 1 mm²×15 μm volume of tissue from the glass slidefrom the tumor region and then from the normal region and place themonto the surface of the paper triangle for paper spray analysis.

A droplet of methanol/water (1:1 v:v; 10 μl) was added to the paper assolvent and then 4.5 kV positive DC voltage applied to produce thespray. Phospholipids such as phosphatidylcholine (PC) and sphingomyelin(SM) were identified in the spectrum (FIG. 17 panels A and B). The peakof [PC(34:1)+K]⁺ at m/z 798 was significantly higher in tumor tissue andpeaks [SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and[SM(36:4)+Na]⁺ at m/z 804 were significantly lower compared with normaltissue.

Example 14: Therapeutic Drug Monitoring

The administration of a drug depends on managing the appropriate dosingguidelines for achievement of a safe and effective outcome. Thisguideline is established during clinical trials where thepharmacokinetics (PK) and pharmacodynamics (PD) of the drug are studied.Clinical trials use PK-PD studies to establish a standard dose, whichmay be fixed or adjusted according formulas using variables like bodymass, body surface area, etc. However, the drug exposure, i.e. theamount of drug circulating over time, is influenced by a number offactors that vary from patient to patient. For example, an individuals'metabolic rate, the type and level of plasma proteins, and pre-existingconditions such as renal and/or hepatic impairment all play a role inaffecting the exposure of the drug in vivo. Further, administration of adrug in combination with other medications may also affect exposure. Asa result, it is often difficult to predict and prescribe an optimumregimen of drug administration.

Over- or underexposure to a drug can lead to toxic effects or decreasedefficacy, respectively. To address these concerns, therapeutic drugmonitoring (TDM) can be employed. TDM is the measurement of active druglevels in the body followed by adjustment of drug dosing or schedules toincrease efficacy and/or decrease toxicity. TDM is indicated when thevariability in the pharmacokinetics of a drug is large relative to thetherapeutic window, and there is an established relationship betweendrug exposure and efficacy and/or toxicity. Another requirement for TDMis that a sufficiently precise and accurate assay for the activeingredient must be available. Immunoassays and liquid chromatographymass spectrometry (LC-MS) are commonly used methods for TDM. Incomparison with immunoassay, LC-MS has advantages which include wideapplicability, high sensitivity, good quantitation, high specificity andhigh throughput. Probes of the invention may be coupled with standardmass spectrometers for providing point-of-care therapeutic drugmonitoring.

The drug Imatinib (GLEEVEC in USA and GLIVEC in Europe/Australia, forthe treatment of chronic myelogenous leu-kemia) in a dried blood spotwas analyzed using paper spray and a lab-scale LTQ mass spectrometer.Quantitation of Imatinib in whole blood was achieved using the MS/MSspectra with a known concentration of Imatinib-d8 being used as theinternal standard (FIG. 14 panel C). The relative response was linearacross a wide range of concentrations, including the entire therapeuticrange (FIG. 14 panel C).

Example 15: High-Throughout Detection

Multiple-tip devices were fabricated and applied for high throughputanalysis (FIG. 28 panel A). The multiple-tip device was a set of papertriangles all connected to a single copper strip (FIG. 28 panel A). Anelectrode was connected to the copper strip. Multiple samples were puton a single paper substrate and analyzed in series using themultiple-tip probe (FIG. 28 panels B-C). Each tip was pre-loaded with0.2 uL methanol/water containing 100 ppm sample (cocaine or caffeine)and dried. Then the whole multiple-tip device was moved on a movingstage from left to right with constant velocity and 7 uL methanol/waterwas applied from the back part for each tip during movement.

To prevent the contaminant during spray, blanks were inserted betweentwo sample tips. FIG. 28 panel (C) shows the signal intensity for thewhole scanning. From total intensity, six tips gave six individual highsignal peaks. For cocaine, peaks only appeared when tip 2 and tip 6 werescanned. For caffeine, the highest peak came from tip 4, which wasconsistent with the sample loading sequence.

Example 16: Tissue Analysis

Direct analysis of chemicals in animal tissue using probes of theinvention was performed as shown in FIG. 29 panel (A). A small sectionsof tissue were removed and placed on a paper triangle. Methanol/water(1:1 v:v; 10 μl) was added to the paper as solvent and then 4.5 kVpositive DC voltage was applied to produce the spray for MS analysis.Protonated hormone ions were observed for porcine adrenal gland tissue(1 mm³, Panel (B)). FIG. 16 is a mass spectrum showing direct analysisof hormones in animal tissue by paper spray. A small piece of pigadrenal gland tissue (1 mm×1 mm×1 mm) was placed onto the paper surface,MeOH/water (1:1 v:v; 10 μl) was added and a voltage applied to the paperto produce a spray. The hormones epinephrine and norepinephrine wereidentified in the spectrum; at high mass the spectrum was dominated byphospolipid signals.

Lipid profiles were obtained for human prostate tissues (1 mm²×15 μm,Panel (C) & (D)) removed from the tumor and adjacent normal regions.Phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM)were identified in the spectra. The peak of [PC(34:1)+K]⁺ at m/z 798 wassignificantly more intense in tumor tissue (panel C) and peaks[SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and [SM(36:4)+Na]⁺at m/z 804 were significantly lower compared with normal tissue (panelD).

Example 17: On-Line Derivatization

For analysis of target analytes which have relatively low ionizationefficiencies and relatively low concentrations in mixtures,derivatization is often necessary to provide adequate sensitivity.On-line derivatization can be implemented by adding reagents into thespray solution, such as methanol/water solutions containing reagentsappropriate for targeted analytes. If the reagents to be used are stableon paper, they can also be added onto the porous material when theprobes are fabricated.

As a demonstration, 5 μL methanol containing 500 ng betaine aldehydechloride was added onto a paper triangle and allowed to dry to fabricatea sample substrate preloaded with a derivatization reagent for theanalysis of cholesterol in serum. On-line charge labeling with betainealdehyde (BA) through its reaction with hydroxyl groups has beendemonstrated previously to be very effective for the identification ofcholesterol in tissue (Wu et al., Anal Chem. 2009, 81:7618-7624). Whenthe paper triangle was used for analysis, 2 μL human serum was spottedonto the paper to form a dried spot and then analyzed by using paperspray ionization. A 10 μL ACN/CHCl₃ (1:1 v:v) solution, instead ofmethanol/water, was used for paper spray to avoid reaction between thebetaine aldehyde and methanol.

The comparison between analysis using a blank and a reagent-preloadedpaper triangle is shown in FIG. 30 panels (A) and (B). Without thederivatization reagent, cholesterol-related peaks, such as protonatedion [Chol+H]⁺ (m/z 387), water loss [Chol+H−H₂O] (m/z 369), and sodiumadduction [Chol+Na]⁺ (m/z 409), were not observed (Panel A). With thederivatization reagent, the ion [Chol+BA]⁺ was observed at m/z 488.6(Panel B). MS/MS analysis was performed for this ion and acharacteristic fragment ion m/z 369 was observed (Panel C).

Example 18: Peptide Pre-Concentration Using Modified Paper SpraySubstrate

Pre-concentration of chemicals on the paper surface using photoresisttreatment. Chromatography paper was rendered hydrophobic by treatmentwith SU-8 photoresist as described previously (Martinez et al., AngewChem Int. Ed., 2007, 46:1318-1320). Then 5 μl bradykinin 2-9 solution(100 ppm in pure H₂O) was applied on the paper surface. When thesolution was dry, the paper was put into water and washed for 10 s.After washing, the paper triangle was held in front of the MS inlet, 10μl pure MeOH was applied as solvent and the voltage was set at 4.5 kVfor paper spray. The same experiment was done with untreated papersubstrate for comparison.

FIG. 31 panel (A) shows the tandem MS spectrum of bradykinin 2-9 frompaper with photoresist treatment. The intensity of the most intensefragment ion 404 is 5.66E3. FIG. 31 panel (B) shows the tandem MSspectrum of bradykinin 2-9 from normal chromatography paper withoutphotoresist treatment. The intensity of the most intense fragment ion404 is only 1.41E1. These data show that the binding affinity betweenphotoresist-treated chromatography paper and peptide is much higher thanthat between normal chromatography paper and peptide, thus more peptidecan be kept on the paper surface after washing by water. When puremethanol is applied, these retained peptides will be desorbed anddetected by MS. This method can be used to pre-concentrate hydrophobicchemicals on the paper surface, and other hydrophilic materials (e.g.salts) can also be removed from the paper surface.

Example 19: Inverted Polarities

The polarity of the voltage applied to the probe need not match thatused in the mass analyzer. In particular, it is possible to operate theprobes of the invention with a negative potential but to record the massspectrum of the resulting positively changed ions. In negative ion mode,a large current of electrons (or solvated electrons) is produced inpaper spray. These electrons, if of suitable energy, can be captured bymolecules with appropriate electron affinities to generate radicalanions.

Alternatively, these electrons might be responsible for electronionization of the analyte to generate the radical cation oralternatively ESI might involve a solvent molecule which might thenundergo charge exchange with the analyte to again generate the radicalcation. If this process occurs with sufficient energy, characteristicfragment ions might be produced provided the radical cation is notcollisionally deactivated before fragmentation can occur.

An experiment was done on a benchtop LTP using toluene vapor, with aprobe of the invention conducted at −4.5 kV with methanol:water assolvent applied to the paper. The spectrum shown in FIG. 32 wasrecorded. One notes that ion/molecule reactions to give the protonatedmolecule, m/z 93 occur as expected at atmospheric pressure. One alsonotes however, the presence of the radical cation, m/z 92 and itscharacteristic fragments at m/z 91 and 65.

An interesting note is that the “EI” fragment ions were most easilyproduced when the source of toluene vapor was placed close to the MSinlet; i.e., in the cathodic region of the discharge between the papertip and MS inlet. This suggests that direct electron ionization byenergetic electrons in the “fall” region might be at least partlyresponsible for this behavior.

Example 20: Cartridge for Blood Analysis

FIG. 33 panel (A) shows an exemplary case for spotting blood onto porousmaterial that will be used for mass spectral analysis. The cartridge canhave a vial with a volume at the center and vials for overflows. A plug,such as a soluble membrane containing a set amount of internal standardchemical, is used to block the bottom of the vial for volume control. Adrop of blood is placed in the vial (Panel B). The volume of the bloodin the vial is controlled by flowing the extra blood into the overflowvials (Panel B). The blood in the vial is subsequently dissolved in themembrane at the bottom, mixing the internal standard chemical into theblood (Panel B). Upon dissolution of the plug, blood flows to the papersubstrate, and eventually forms a dried blood spot having a controlledamount of sample and internal standard (Panel B).

Example 21: Ion Generation Using Wetted Porous Material and a DryingAgent

a. Solvent Applied to Liquid Blood on a Porous Substrate and Dried Bloodon a Porous Substrate

Systems and methods described herein use anhydrous salts (drying agents)to dry and thus restrict the flow of liquid blood during paper sprayanalysis. FIG. 37 panel (A) shows result of applying solvent too early(<2 hours) to blood spot on paper. The mixture of blood and solventcauses the blood to run through the paper and decreases the efficiencyof the paper spray. FIG. 37 panel (B) shows systems and methods in whichthe porous substrate is pre-loaded with a drying agent before spottingthe blood, allowing for more rapid analysis.

b. Drying Agent for Rapid Analysis of Whole Blood

A drying agent, such as magnesium sulfate, sodium sulfate, sodiumcarbonate, or calcium chloride is mixed and pre-spotted along with theinternal standard, thus saving a sample preparation step. Anotherpotential agent, is a blood coagulant such as alum powder. As is seen inFIG. 38 and in Table 1 below, the method of pre-spotting the internalstandard and drying agent provided a very respectable limit of detectionwith significantly reduced drying times.

TABLE 1 RSD LOD Drying Method IS Method RSD blank normal signal (ng/mL)2 Hours Air Mixed 32% 4% 18 MgSO₄ Pre-spotted 55% 36% 56

Further improvements include (i) drying/crystallizing the drying agenton the paper; (ii) drying agent choice; (iii) Compatibility with papertype, e.g. thickness & pore size; (iv) combination of a drying agent andoven drying.

In certain embodiments, a desiccator may be used to store the pre-loadedpaper in a moisture-free atmosphere. Such a desiccator could be anycontainer that contains the commercially-available Drierite. This schemeis shown in FIG. 39.

Example 22: Blood Coagulant as a Drying Agent

Paper spray mass spectrometry is applied to oncology drugs in freshwhole blood samples supported on filter paper substrates instead of dryblood as done previously. Addition of the coagulant alum clotted theblood and allowed for immediate sample analysis. The coagulant did notinterfere with the function of the paper spray nor did it add featuresto the mass spectra. Quantitative analysis of therapeutic drugs in theblood was achieved utilizing internal standards which were pre-spottedonto the filter paper. Eight oncology drugs were examined, with lowerlimits of detection ranging between 0.5 and 17 ng/mL and linear dynamicsranges greater than two orders of magnitude. Inter-day accuracies ofquality controls for pazopanib ranged from 102 to 118%, withimprecisions of 9 to 13%. This one-step method was conducted using 10 μLof blood, a drop of solvent, and took about 45 seconds per trial. Theseresults indicate applicability to point-of-care therapeutic drugmonitoring in a clinical setting.

a. Introduction

Every drug has an optimally effective therapeutic range, outside ofwhich the drug is harmful or ineffective. Concentrations of activepharmaceutical compounds in a patient's circulatory system vary with theindividual (A. Y. H. Lu, Drug Metab. Dispos., 1998, 26, 1217-1222). Oneprocedure for determining the upper limit is to increase drug dosageuntil dose-limiting toxicity occurs. Alternative approaches includepharmacogenomics (W. E. Evans and M. V. Relling, Science, 1999, 286,487-491; W. E. Evans and H. L. McLeod, N. Engl. J. Med., 2003, 348,538-549; and W. E. Evans and M. V. Relling, Nature, 2004, 429, 464-468)and pharmacokinetic modeling (N. H. G. Holford and L. B. Sheiner, ClinPharmacokinet, 1981, 6, 429-453; and L. E. Gerlowski and R. K. Jain, J.Pharm. Sci., 1983, 72, 1103-1127) which use genetics and statistics toaccount for the variation in uptake and metabolism of drugs. Anotherpossibility is therapeutic drug monitoring (TDM), in which drug exposurelevels within the body are measured directly by a quantitative method.Historically, immunassays and high performance liquid chromatographywith optical or mass spectrometric detection have been themethods-of-choice for therapeutic drug monitoring. Immunoassays havebeen used for decades for point-of-care monitoring of blood, and recentresearch has provided faster results for point-of-care applications (X.Yang, J. Janatova, J. M. Juenke, G. A. McMillin and J. D. Andrade, Anal.Biochem., 2007, 365, 222-229; and T. Tachi, T. Hase, Y. Okamoto, N.Kaji, T. Arima, H. Matsumoto, M. Kondo, M. Tokeshi, Y. Hasegawa and Y.Baba, Anal. Bioanal. Chem., 2011, 401, 2301-2305) by implementingmicrofluidic methods (B. Zheng and R. F. Ismagilov, Angew. Chem. Int.Ed., 2005, 44, 2520-2523). The ultimate goal for TDM is individualizeddosing during treatment using a platform technology for fast, accurate,and inexpensive assays directly from untreated biofluid.

This Example shows the use of coagulants for rapid quantitative analysisof oncology drugs from fresh whole blood by paper spray massspectrometry. Oncology drug analysis has been chosen as a model problemfor point-of-care analysis due to the need for fast results duringchemotherapy. The quantitative results herein show that paper spray hassufficient sensitivity and precision, with regard to clinicalregulations, to provide the first one-step analysis of fresh whole bloodfor applications in point-of-care mass spectrometry.

b. Materials

Most drug standards were purchased from Sigma-Aldrich (St. Louis, Mo.,USA) and used without any further purification. All organic solvents(HPLC grade) were purchased from VWR Scientific (Chicago, Ill., USA).Blood card paper (grade 31 ETF) was obtained as a sample from Whatman(Piscataway, N.J., USA). Bovine whole blood with potassium ethylenediamine tetraacetic acid (K₂EDTA) was purchased from Innovative Research(Novi, Mich., USA) and stored at 4° C.

c. Preparation of Samples

All stock solutions were prepared in methanol, and working solutionswere prepared in 50:50 methanol/water. Spiked blood samples wereprepared by adding the necessary quantity of working solution not toexceed 5% of the volume of blood (typically, addition of 50 □L 20×concentration solution to 950 □L of bovine whole blood). Before samplepreparation, the blood was incubated to a temperature of 37° C. Theblood standards were vortexed for 10 seconds and analyzed within 1 hourof preparation.

d. Mass Spectrometry and Paper Spray Ionization

FIG. 40 is a schematic showing the mode of operation for point-of-caretherapeutic drug monitoring of whole blood by paper spray massspectrometry utilizing a coagulating agent. Each sheet of Whatman 31ETFpaper had a grid drawn with pencil, each pixel of which was 1 cm×2 cm insize (FIG. 40). A mixture of internal standard (500 ng/mL) and alumpowder (60 mg/mL), dissolved in distilled water, was pipetted (5 μL) atthe center of each of the marked grid boxes. After allowing the spots todry in air for approximately 10 minutes, the pre-spotted paper sheet wascut into small triangles, measuring 8 mm at the base and 15 mm inheight. The center of each triangle contained the pre-spotted alum andinternal standard solution.

For blood sample analysis, the back edge of the paper triangle was heldby a toothless copper alligator clip which was connected to the highvoltage power supply (FIG. 40). The front tip of the paper was placed 5mm away from the atmospheric pressure inlet of the mass spectrometer.The whole blood sample was then pipetted (10 μL) onto the center of thepaper triangle. Pure methanol (or methanol with 0.1% sodium acetate fordrugs that formed sodiated adducts) was applied slowly to the back endof the paper, behind the blood spot, until the solvent wicked throughthe blood and to the front tip of the triangle. The applied spraysolvent was only enough to wet the entire blood spot and paper triangleand did not exceed 32 μL for each spray process. Since the solvent flowsfrom the back to front tip, if not enough solvent is applied, no spraywill occur. On the other hand, if too much solvent is applied, 1) thesolvent may travel over the blood spot instead of through it, whichreduces the analyte pick-up; 2) the solvent flow rate at the spray tipwill be too high and cause the paper to eject very large droplets whichdo not evaporate properly. Following addition of the solvent, 3000 V wasapplied to the paper via the alligator clip, producing a spray currentbetween 0.2 and 0.6 μA. The paper spray procedure left the blood spotstationary and intact, thus reinjection analysis was performed byapplying solvent and a high voltage on the same sample for a secondtime.

Dried blood spot analysis was performed in the same fashion as freshblood, but without the coagulant and with an extra two hours for blooddrying, in accordance to the procedure of Manicke et al. (J. Am. Soc.Mass. Spectrom., 2011, 22, 1501-1507). All experiments were performedusing a TSQ Quantum Access MAX (Thermo Scientific, San Jose, Calif.,USA). Data collection was executed in selected reaction monitoring (SRM)mode (R. W. Kondrat and R. G. Cooks, Anal. Chem., 1978, 50, A81-A92),observing the desired fragment intensity for the drugs and internalstandards by collision-induced dissociation (CID). Scan times were 250ms, alternating between the drug and internal standard, and data werecollected until the spray voltage was manually turned off at 15 seconds,totaling 30 scans for each fragment ion.

e. Data Analysis

The two sets of 30 scans were plotted as individual ion chronograms,showing ion current versus time for each fragment ion. The area underthe curve corresponding to the drug was compared to that of the internalstandard and used for quantitation. Limits of detection were determinedusing a calibration curve consisting of four trials each of 0, 50, 100,500, and 1000 ng/mL drug standard solutions. Limits of detection weredetermined by multiplying the t-value (95% confidence) by the standarddeviation of the blank, divided by the slope of the calibration curvedetermined by 1/x² weighted linear least squares (G. L. Long and J. D.Winefordner, Anal. Chem., 1983, 55, A712-A724). Reported precisionvalues are the relative standard deviation of concentration determinedfrom multiple blood spots taken from a single blood sample. Accuracy isdefined to be the measured concentration divided by the actualconcentration multiplied by 100%.

f. Results

One of the primary challenges faced by rapid blood analysis with paperspray is the contamination of the spray tip by blood components. In thecase of freshly-spotted blood that was not dried, oxygenatedhemoglobin—the red component of blood—was seen to run to the tip (seeFIG. 41). This phenomenon, which presumably indicated the movement ofother proteins and cellular components toward the tip as well,prohibited formation of a stable Taylor cone using volumes of bloodgreater than 2 μL. Lower volumes of blood (<2 μL) can be analyzed in afew minutes (H. Wang, J. J. Liu, R. G. Cooks and Z. Ouyang, Angew. Chem.Int. Ed., 2010, 49, 877-880) but are less desirable due to poorer limitsof detection and increased standard deviations. One requirement of aviable bioanalytical blood analysis method by paper spray, therefore, isthat the red blood cells, proteins, and other sources of interferenceremain stationary on the paper. While this fixation occurs naturally inthe case when dried blood spots are extracted with organic solvents,additional means are needed for freshly spotted blood.

One approach for the rapid paper spray analysis of fresh blood sampleswas the addition of coagulants to the paper. A pre-spotted coagulant,potash alum, quickly clotted the blood (within seconds) and allowed thespray solvent to wick through the blood spot and to the tip of thepaper, dissolving analytes in the process (FIG. 41). Potash alum, morespecifically potassium aluminum sulfate [KNH₄(SO₄)₂.H₂O], is a commonproduct found in powdered alum which promotes flocculation in suspendedcolloids. Potash alum is also minimally soluble in methanol (a commonand effective paper spray solvent) and does not produce any contaminantions during mass spectrometry. Alum was the coagulant used in all of thefollowing experiments.

g. Therapeutic Drug Monitoring

Two important parameters characterizing the method are precision andaccuracy. The imprecision should not exceed a relative standarddeviation of 15% (20% at the lower limit of quantitation). Theinaccuracy of quality control samples likewise should not exceed ±15%(±20% at the lower limit of quantitation). Table 2 is a representativequality control table for analysis of pazopanib in blood, portraying theinter- and intra-day accuracy and reproducibility of the oncology drugassay (n=5 for each concentration, each day). Pazopanib is ananti-cancer drug which has been approved by the U.S. FDA.

The quality control results from Table 2 suggest that this method is aviable bioanalytical method for point-of-care therapeutic drugmonitoring. Most of the quality control samples yielded a positive bias,which was most likely due to error in the sample preparation process.All of the inter-day percent accuracies and standard deviations for thisdata set produced errors under 15%, on average 10.8%. This is comparableto previous work on dried blood spots by paper spray with pre-spottedinternal standards, which had about 10% relative standard deviations (N.E. Manicke, P. Abu-Rabie, N. Spooner, Z. Ouyang and R. G. Cooks, J. Am.Soc. Mass. Spectrom., 2011, 22, 1501-1507). The most unusual result,from 750 ng/mL on Day 1, seems especially erroneous and was most likelycaused by experimental errors during the paper spray process.

TABLE 2 Quality control of pazopanib by paper spray analysis of 10 μLfresh whole blood Pazopanib Mean % Accuracy {Imprecision (% CV)}concentration Day 1 Day 2 Day 3 Inter-day 15 ng/mL 113.0% {12.3%} 110.9%{12.6%} 129.3% {8.7%} 117.7% {11.2%} (3 × LLOQ) 50 ng/mL  97.1% {13.5%}101.2% {3.9%} 106.4% {10.8%} 101.6% {9.4%} 500 ng/mL  107.7% {9.5%}111.6% {12.9%} 103.7% {8.9%} 107.6% {10.4%} 750 ng/mL  116.3% {16.5%}102.9% {7.5%} 101.2% {12.4%} 106.8% {12.1%}

It is conceivable that in the course of point-of-care work, reanalysisof a sample will be needed due to instrument malfunction or requiredimmediate result confirmation. Therefore, reinjection reproducibilitywas analyzed in which the solvent was reapplied and paper spray wasperformed on the same blood spot for a second time. In general, paperspray does not have high recovery of the analyte. That is, the amount ofanalyte extracted and sprayed from the paper toward the massspectrometer is only a portion of the available analyte (Z. Zhang, W.Xu, N. E. Manicke, R. G. Cooks, Z. Ouyang, Anal. Chem., 2012, DOI:10.1021/ac202058w). Although the relatively lower recovery contributedto poorer sensitivity, it did allow for reanalysis of the same bloodspot. Table 3 shows how analyzing the same blood sample a second timedid not affect the accuracy of the results, with the exception of the 10ng/mL samples, the concentrations of which were less than three timesthe lower limit of quantitation.

TABLE 3 Reinjection of pazopanib standards from fresh blood paper spraymass spectrometry Pazopanib Mean Drug/IS (n = 6) Concentration Injection1 Injection 2 % Change Blank 0.0087 0.0084 3.4%  10 ng/mL 0.057 0.06717.5%  50 ng/mL 0.253 0.251 0.8% 100 ng/mL 0.522 0.506 3.1% 500 ng/mL3.114 3.029 2.7% 1000 ng/mL  5.713 5.754 0.7%

It should be noted that the parameters for paper spray were chosencarefully for optimal performance. To achieve precision of better than15%, it was necessary to use an isotopically-labeled internal standarddue to the various affinities of the analytes for the substrate, as wellas inefficiencies in ionization and ion transfer. In addition, althoughvolumes of blood less than 5 μL clotted more efficiently, 10 μL of bloodwas typically used in order to improve the standard deviation of themethod. It was difficult, for example, to control the blood volume, spotsize, and spot shape for smaller volumes of micro-pipetted blood. Anautomated technique would allow for smaller blood volumes to be analyzedmore reproducibly. The spray solvent, methanol, was chosen to achieveproper wicking through the paper, good analyte extraction, and lowbackground noise. The background noise was reduced by using the lowestspray voltage possible, typically 3.0 kV.

FIG. 42 shows six consecutive trials (from different blood spots) ofpazopanib. The method of pre-spotting the clotting agent and theinternal standard resulted in a relative standard deviation of 9 to 12%.A key feature of the chronograms was that although the internal standardwas pre-spotted, the peak shapes of the drug and the internal standardwere similar, resulting in acceptable reproducibility.

A wide variety of oncology drugs was explored by the same method. Thesedrugs varied in molecular weight from 261 g/mol (cyclophosphamide) to854 g/mol (paclitaxel). There are many factors that affected the lowerlimits of detection for these drugs, including solubility in the spraysolvent, affinity for the substrate and blood components, protonaffinity (or Na⁺ affinity in the case of the taxanes which form sodiumion adducts), background noise level, and selectivity of the SRMtransition. A previous study demonstrated that matrix effects causenegligible analyte suppression within the therapeutic range for drugs bypaper spray, and that paper spray MS measures the total drugconcentration in blood (free plus bound drug) (N. E. Manicke, Q. A.Yang, H. Wang, S. Oradu, Z. Ouyang and R. G. Cooks, Int. J. Massspectrom., 2011, 300, 123-129).

Oncology drugs pazopanib, tamoxifen, imatinib, cyclophosphamide,paclitaxel, docetaxel, topotecan, and irinotecan were each analyzedindividually to determine lower limits of detections (LLOD's). As Table4 shows, the method described herein for rapid drug analysis from freshblood gave LLOD's ranging from 0.5 ng/mL for pazopanib, to 17 ng/mL fortopotecan; all these values are in the same order of magnitude as thedried blood spot experiments by paper spray (N. E. Manicke, P.Abu-Rabie, N. Spooner, Z. Ouyang and R. G. Cooks, J. Am. Soc. Mass.Spectrom., 2011, 22, 1501-1507). The LLOD's were within one order ofmagnitude compared to previously-reported HPLC-MS/MS methods oftherapeutic drugs.

TABLE 4 Limits of detection for oncology chemotherapeutic drugs by paperspray mass spectrometry 15 μL Dried 10 μL Fresh Therapeutic PrecursorFragment Internal IS IS Blood Spot Blood w/Alum Range Ion Ion StandardPrecursor Fragment LLOD LLOD Drug (ng/mL)^(a) (m/z) (m/z) (IS) Ion Ion(ng/mL) (ng/mL) Pazopanib NA 438.1 357.1 ²H₃ ¹³C- 442.1 361.1 1 0.5 [M +H]⁺ pazopanib [M + H]⁺ Tamoxifen 35-45 372.2 72.2 ¹³C₂ ¹⁵N- 375.2 75.213 8 [M + H]⁺ tamoxifen [M + H]⁺ Imatinib  900-1800 494.2 394.0imatinib-d8 502.2 394.0 1.2 9 [M + H]⁺ [M + H]⁺ Cyclo- 10000-25000 261.0140.0 Ifosfamide 261.0 154.0 13 11 phosphamide [M + H]⁺ [M + H]⁺Paclitaxel  85-1000 876.5 307.9 Docetaxel 830.3 549.2 15 12  [M + Na]⁺ [M + Na]⁺ ^(a)Therapeutic Ranges for plasma concentrations (R.Regenthal, M. Krueger, C. Koeppel and R. Preiss, J. Clin. Monitor Comp.,1999, 15, 529-544.)

When analyzing fresh blood, it was hypothesized that any biomoleculesthat are soluble in the spray solvent and ionizable by paper spray willbe detected by the mass spectrometer. FIG. 43 shows that in addition tothe drugs that were spiked into the blood, free heme was observed, aswell as some common phospholipids. The presence of heme indicates thatred blood cell lysis is occurring due to the solvent composition and/orpaper spray conditions. It is reasonable to expect that futureexperiments may involve the analysis of lipids, proteins, and/or otherbiomolecules which would also be useful for point-of-care clinicaldiagnostics.

h. Conclusion

Quantitative analysis of oncology drugs by paper spray with alum, acoagulant, shows that this method has capabilities for point-of-caretherapeutic drug monitoring. Utilizing a bench-top mass spectrometer,inter- and intra-day percent accuracies and imprecisions of qualitycontrols and standards were within regulated guidelines (<15% error)using pre-spotted isotopically-labeled internal standards. The time spanfrom blood spotting to results took under 60 seconds, due to the onlineextraction of paper spray and the coagulating ability of pre-spottedalum. The excellent limits of detection suggest that this method shouldbe easily transferrable to a portable mass spectrometer where the largerblood volumes that can be handled quickly with the drying agent will benecessary for adequate analytical performance. The use of inexpensiveexpendables means that disposable cartridges could be used which wouldreduce sample contact and lower the potential risk forcross-contamination.

This method could also find a place in home-use applications, forexample in an assisted living facility to ensure day-to-day medicationlevels. The small blood consumption by paper spray, several microliters,would also allow co-monitoring of blood by separate aliquots andanalyzing electrolytes, blood gases, and glucose levels. In addition totherapeutic oncology drugs and metabolites, paper spray may be appliedfor analysis of biocompounds such as fatty acids, lipids, and proteins,as well as other applications including drugs of abuse, steroids, andhuman growth hormones.

Example 23: Ion Generation Using a Wetted Porous Substrate thatSubstantially Prevents Diffusion of Sample into the Substrate

a. Analysis of Whole Blood

When fresh (liquid) blood is dropped onto sample substrates of lesshydrophobicity, such as print paper or Silanized paper, the blood dropstays on the top of the substrate without diffusing into the poroussubstrate (FIG. 44A). When a solvent of proper hydrophobicity is droppedon the substrate with the blood droplet, the blood defuses with thesolvent (FIG. 44B).

The solvent can extract the chemicals from the blood and also facilitatethe spray to generate ions from the substrate (FIG. 45). Certainchemicals could be selectively extracted from the whole blood while mostother chemicals stay on the paper substrate reducing the matrix effectand contamination to the mass spectrometer. When a high voltage isapplied onto the paper triangle, the charged droplets containing thechemicals will be sprayed from the tip of the triangle, as shown in FIG.45.

b. Direct Detection of Proteins from Whole Fresh Blood

Even though there is a large amount of proteins in whole blood, it isdifficult to detect any protein spectra from a dried blood spot usingchromatography paper. Without being limited by any particular theory ormechanism of action, it is believed that when blood is dried on paper,the proteins in the blood could be denatured and combine strongly withthe substrate especially if the substrate is polar.

10 μL of bovine whole blood was dropped onto a paper surface. Then 50 μLmethanol with 1% acetic acid was applied as solvent. 4 kV DC voltage wasapplied on the copper clip to produce spray. FIG. 46 shows that proteinspectra could be detected from all the three paper substrates when freshwhole blood was used, especially for silanized paper which is quitehydrophobic. α and β subunits of hemoglobin could be detected as thedominated peaks in the spectra. The peak at m/z 616 represents the hemegroup from the hemoglobin. When chromatography paper and printer paperwere used, the spectra were dominated by lipid peaks and the proteinpeaks have a low relative intensity. When silanized paper substrate wasused, there is intense hemoglobin signal. The two sets of proteinspectra in FIG. 46C represents α and β subunits of hemoglobin,respectively. The spectra was recorded using an Exactive Orbitrap massspectrometer.

c. Extraction of Chemicals from Fresh Whole Blood Using Organic Solventsthat are not Miscible with Blood

FIG. 47A shows quantitation of cotinine, the major metabolite ofnicotine, from fresh bovine whole blood. Printer paper was used as thesubstrate and 10 μL of bovine whole blood was dropped onto the papersurface. Then 30 μL 90% dichloromethane and 10% isopropanol was appliedas solvent, which has been shown to be an effective solvent to extractcotinine from whole blood (F. Baumann, R. Regenthal, I. L.Burgos-Guerrero, U. Hegerl, R. Preiss, J Chromatogr B878, 107 (Jan. 1,2010)). 4 kV DC voltage was applied on the copper clip to produce spray.The calibration curve was obtained by spiking different concentrationsof cotinine into the blood sample and 100 ppb cotinine-d3 was used asinternal standard for each sample. LOQ of 1 ppb could be reached. Thespectra were recorded using TSQ mass spectrometer. As shown in FIG. 47,printer paper was used since it was less hydrophilic compared with Grade1 chromotography paper.

Most of the blood sample stayed on top of the paper substrate forming afilm of liquid blood instead of being absorbed into the paper. When thesolvent was applied, it went through the blood sample to the tip. Whenhigh voltage is applied, a strong spray plum was produced from the tipof the paper triangle (FIG. 47B). During the whole process, the bloodsample stayed in the original position. Since the solvent was notmiscible with blood sample, the analytes in the blood were extractedthrough a liquid-liquid extraction process.

d. Extraction of Chemicals from Fresh Whole Blood Using Organic Solventswhich are Miscible with Blood

FIG. 48A shows quantitation of nicotine from fresh bovine whole blood.Print paper was used as the substrate and 10 μL of bovine whole bloodwas dropped onto the paper surface. Then acetonitrile was applied assolvent to extract nicotine from fresh whole blood. 4 kV DC voltage wasapplied on the copper clip to produce spray. The calibration curve wasobtained by spiking different concentrations of nicotine into the bloodsample and 100 ppb nicotine-d3 was used as internal standard for eachsample. LOQ of 0.1 ppb was reached. The spectra were recorded using TSQmass spectrometer. Printer paper was used since it was less hydrophiliccompared with Grade 1 chromotography paper.

When high voltage was applied, a strong spray plum was produced from thetip of the paper triangle (FIG. 48B). Even though acetonitrile ismiscible with water in blood, most of the the blood sample still staysin the original position during the spray.

e. Applying Sample Extraction to Make Dried Sample Spots for LaterAnalysis

L whole fresh blood was spotted on to the print paper. Then 10 μLacetonitrile was applied onto the blood sample before it dried toextract nicotine from the blood sample. After the sample and solventwere dried on the paper, another 20 μL acetonitrile was applied as spraysolvent and 4 kV DC was applied to generate paper spray. LOQ of 1 ppbcould be achieved. The spectra were recorded using TSQ mass spectrometer(FIG. 49).

Example 24: Analysis of Tobacco Nicotine Alkaloids from Biofluids Usinga Wetted Porous Substrate that Substantially Prevents Diffusion ofSample into the Substrate

The determination of tobacco nicotine alkaloids from biofluids is ofgreat importance for a smoking test, to tobacco cessation treatment, andto the study of exposure to secondhand smoke and effect of tobacco useon individual health. Paper spray mass spectrometry has been developedfor direct, quantitative analysis of tobacco nicotine alkaloids fromdried and liquid biofluids such as blood, urine and saliva. Limit ofquantitation as low as several ng/mL were obtained for nicotine,cotinine, trans-3′-hydroxycotinine and anabasine. Due to the fast andconvenient characteristics of this method, it shows potential forsignificantly improving the analytical efficiency in clinical labs andalso for point-of-care tobacco use assessment.

a. Introduction

Tobacco use is the chief preventable cause of disease and death in theUS (Centers for Disease Control and Prevention. AnnualSmoking-Attributable Mortality, Years of Potential Life Lost, andEconomic Costs—United States, 1995-1999. Morbidity and Mortality WeeklyReport 2002; 51(14):300-3). However, due to it addiction nature, to quitsmoking is extremely difficult. Based on the data from CDC, in 2010,about 19.3% of all adults are smokers in the US (Centers for DiseaseControl and Prevention. Vital Signs: Current Cigarette Smoking AmongAdults Aged ≥18 Years—United States, 2005-2010. Morbidity and MortalityWeekly Report 2011; 60(33):1207-12) and only less than 10 percent whoquit smoking for a day remain abstinent one year later (M. C. Fiore,Med. Clin. N. Am. 1992, 76, 289-303). Smokers have much higher healthrisks for a series of serious diseases, such as coronary heart disease,stroke, and lung cancer. The adverse health effects of smoking accountsfor about one fifth deaths each year (Centers for Disease Control andPrevention. Annual Smoking-Attributable Mortality, Years of PotentialLife Lost, and Economic Costs-United States, 1995-1999. Morbidity andMortality Weekly Report 2002; 51(14):300-3) and $193 billion were spentannually due to cigarette smoking caused health-related economic lossesin the US (Centers for Disease Control and Prevention.Smoking-Attributable Mortality, Years of Potential Life Lost, andProductivity Losses-United States, 2000-2004. Morbidity and MortalityWeekly Report 2008; 57(45):1226-8).

Because of the significant adverse health effects of tobacco use, moreand more insurance companies request life insurance buyers to do smokingtests. Different rating categories will be given based on the smokingstatus. Many employers also use smoking testing to evaluate prospectiveemployees for tobacco use.

For tobacco use assessment and other tobacco use related clinicaldiagnosis, nicotine and its metabolites are most frequently analyzedchemicals (I. Kim, M. A. Huestis, J. Mass Spectrom. 2006, 41, 815-821;and J. Hukkanen, P. Jacob, N. L. Benowitz, Pharmacol. Rev. 2005, 57,79-115). Nicotine is the major addictive chemical in tobacco which makespeople difficult to quit smoking once started (N. L. Benowitz, Annu.Rev. Pharmacol. Toxicol. 2009, 49, 57-71; and N. L. Benowitz, N. Engl.J. Med. 2010, 362, 2295-2303). Nicotine is also the major component inthe pharmacotherapy for smoking cessation (J. E. Henningfield, N. Engl.J. Med. 1995, 333, 1196-1203). To reduce the withdrawal symptoms duringsmoking cessation, nicotine medications such as nicotine gum are oftenutilized which provide lower, and more stable blood nicotineconcentration.

Nicotine is mainly metabolized in the liver by an enzyme CYP2A6 (J.Hukkanen, P. Jacob, N. L. Benowitz, Pharmacol. Rev. 2005, 57, 79-115).The half-life of nicotine is relatively short (about 2 hours), thus theassessment of nicotine in biofluids cannot reflect the status of tobaccouse accurately. Cotinine is the major metabolite of nicotine with a muchlonger half-life (about 16 hours). Because of the long half-life,cotinine is being used as the biomarker for cigarette smoking andenvironmental tobacco smoke exposure. Cotinine is further metabolized bythe same enzyme CYP2A6 to trans-3′-hydroxycotinine (3HC). Recent studyshowed that the ratio of 3HC to cotinine could provide a convenientmeasure to phenotype individuals for CYP2A6 activity which is abiomarker of nicotine metabolism guiding dosage in nicotine medication(N. L. Benowitz, O. F. Pomerleau, C. S. Pomerleau, P. Jacob, Nicotine &Tobacco Research 2003, 5, 621-624; P. Jacob, L. S. Yu, M. J. Duan, L.Ramos, O. Yturralde, N. L. Benowitz, Journal of ChromatographyB-Analytical Technologies in the Biomedical and Life Sciences 2011, 879,267-276; and D. Dempsey, P. Tutka, P. Jacob, F. Allen, K. Schoedel, R.F. Tyndale, N. L. Benowitz, Clin. Pharmacol. Ther. 2004, 76, 64-72).Although nicotine and its metabolites are widely used for tobacco useassessment, they cannot be used to distinguish smokers from personstaking nicotine medications. In such a condition, another tobaccoalkaloid anabasine can be used which only exists in tobacco but not innicotine-medication products (P. Jacob, L. Yu, A. T. Shulgin, N. L.Benowitz, Am. J. Public Health 1999, 89, 731-736; and P. Jacob, D.Hatsukami, H. Severson, S. Hall, L. Yu, N. L. Benowitz, CancerEpidemiol. Biomark. Prev. 2002, 11, 1668-1673).

This Example shows the power of this technique for direct quantitationof biomarkers of tobacco use from biofluids, including blood, urine andsaliva. Besides the testing them in dried spots, the Example alsodemonstrates the direct analysis of fresh liquid samples.

b. Materials

Bovine whole blood was purchased from Innovative Research (Novi, Mich.).Synthetic urine sample was purchase from CST Technologies (Great Neck,N.Y.). Saliva sample was donated by Dr. Ouyang who is the PI of thisstudy.

Nicotine, cotinine, and anabasine were purchased from Sigma-Aldrich (St.Louis, Mo.). Trans-3′-hydroxycotinine and trans-3′-hydroxycotinine-d3were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).Nicotine-d3 and cotinine-d3 were purchased from CDN Isotopes (Quebec,Canada). Anabasine-d4 was purchased from United States Biological(Swampscott, Mass.).

All experiments were performed using a TSQ Quantum Access Max massspectrometer (Thermo Scientific, San Jose, Calif.). All analytes weremonitored in the selected reaction monitoring (SRM) mode. The SRMtransitions are shown in Table 5 below.

TABLE 5 SRM transitions Analyte Parent ion m/z Fragment ion m/z nicotine163, (M + H)⁺ 130 nicotine-d3 166, (M + H)⁺ 130 cotinine 177, (M + H)⁺80 cotinine-d3 180, (M + H)⁺ 80 trans-3′-hydroxycotinine 193, (M + H)⁺80 trans-3′-hydroxycotinine-d3 196, (M + H)⁺ 80 anabasine 163, (M + H)⁺118 anabasine-d4 167, (M + H)⁺ 122

To get the calibration curves, bovine whole blood was spiked with eachchemical and corresponding isotope labeled internal standard. For driedblood spot analysis, the paper was loaded with 5 μL whole blood, placedin the open air overnight, and then stored in a sealed plastic bagcontaining desiccant at room temperature.

Grade 31ET chromatography paper was purchased from Whatman (Piscataway,N.J.). Printer paper (letter size, 75 g/m2) was purchased from Xerox(Norwalk, Conn.).

For this Example, the paper was cut into a triangle (base 7 mm, height12 mm). A copper clip was used to hold the paper triangle in front ofthe mass spectrometer. 4 kV DC high voltage was applied through thecopper clip to induce paper spray ionization from tip of the papertriangle. Unless otherwise noted, 90% acetonitrile with 10% water wasused as the spray solvent.

For the calibration curves, 3 samples were repeated at eachconcentration. The lower limit of quantitation (LLOQ) was calculated as10 times of standard deviation of blank samples only with internalstandards divided by the slope of the calibration curve.

c. Results

Nicotine, cotinine, 3HC, and anabasine (FIG. 50) were tested directlyfrom dried blood spots using paper spray ionization. The calibrationcurves of the four chemicals are shown in FIG. 51. Good linarites wereobtained throughout the whole concentration range tested. The LOQs ofthese four chemicals are between 1˜3 ng/mL. As one of the major tobaccouse biomarkers, the cut-off value of cotinine is ˜14 ng/mL betweensmokers and non-smokers (M. J. Jarvis, H. Tunstallpedoe, C. Feyerabend,C. Vesey, Y. Saloojee, Am. J. Public Health 1987, 77, 1435-1438).

Different paper substrates were tested trying to minimize the matrixinterference from the paper substrates and improve the LOQ.Chromatography paper with different thickness (grade 1 0.18 mm and 31 ET0.5 mm), silica coated paper, and printer paper were tested.Interestingly, it was found that printer paper was one of the best papersubstrates for these four chemicals. For 3HC and cotinine, similarresults were obtained using chromatography paper and printer paper.However, for nicotine and anabasine, printer paper provided much lowerbackground signal than chromatography paper thus significantly improvedthe LOQ. It was found that when organic solvent was continuously appliedon printer paper, the background was low for the first several minutes,and then the background will gradually increase. It was observed thatorganic solvent are more difficult to penetrate through the papersubstrate to the reverse side of the paper. These observations suggestthat the lower background from printer paper may be because matrixchemicals were more slowly extracted from the printer paper substrate.The slow extraction of matrix chemicals may be due to the smaller poresize of the printer paper.

For point-of-care test, people may want to get the results as fast aspossible. Even though the analysis of dried blood spot using paper spraycan be done within one minute, the time to allow the blood dry on thepaper can take hours. Addition of the coagulant alum has been shown toallow paper spray analysis before the blood is dried. See Examplesabove. Here, paper spray was also tested for direct analysis of liquidblood samples without any additive. As shown in FIGS. 52A-B, 5 μL freshbovine blood was added onto the printer paper and smeared by a pipettetip forming a thin blood film. Then 20 μL acetonitrile was addedimmediately and 3.5 kV DC high voltage was applied. It was observed thatthe spray plume was produced from the tip. It was interesting to seethat during the spray the blood cells with red color were still trappedin their original position and would not contaminate the instrument(FIGS. 52B-D). It was observed that no protein spectra were obtainedduring the spray of fresh blood sample from the cellulose based papersubstrates which could be due to the strong combination of the proteinmolecules to the paper substrates. However, by changing thehydrophobicity of the paper substrate, protein spectra could also beobserved (FIG. 53). Different concentrations of nicotine and itsisotope-labeled internal standard were spiked into bovine blood to getthe calibration curve from the fresh blood. The LOQ of nicotine in thisway was even better than dried blood spot, which is 0.1 ppb.

Besides blood, cotinine was also tested directly from fresh liquidsaliva and urine. 5 μL saliva sample was added onto Grade 1chromatography paper, and then 30 μL acetonitrile was applied assolvent. The calibration curve is shown in FIG. 54. The LOQ was 2 ng/mLwhich is lower than its cut-off values 14 ng/mL (M. J. Jarvis, H.Tunstallpedoe, C. Feyerabend, C. Vesey, Y. Saloojee, Am. J. PublicHealth 1987, 77, 1435-1438). For the urine sample, it was found that thematrix effect was more serious compared with the blood and saliva sampledue to its high concentration of metal salts. No cotinine could bedetected under 30 ng/mL using acetonitrile as the solvent. It has beenfound that the addition of high levels of ammonium acetate cansignificantly counteract the signal suppression of protein solutionscaused by metal ions (A. T. lavarone, O. A. Udekwu, E. R. Williams,Anal. Chem. 2004, 76, 3944-3950). This is presumably because of theprecipitation of metal ions from solution within the evaporatingelectrospray droplets. Similar improvement was found herein. By adding20 mM ammonium acetate into the urine solution, the LOQ could reach aslow as 5 ng/mL with the cut-off value to be 50 ng/mL.

d. Conclusion

Data herein show direct quantitation of tobacco nicotine alkaloids frombiological fluids in the form of both dried spots and fresh liquid. Thismethod shows potential in quantitating human exposure to tobacco and instudying the nicotine metabolism.

Example 25: Ion Generation Using a Wetted Porous Substrate in which atLeast a Portion of the Porous Substrate Includes a Material thatModifies an Interaction Between a Sample and the Substrate

In this Example, paper spray was coupled with a commercial TSQ and ahome-made MINI 11 for analysis of different target drugs in dried bloodspots, respectively. Different from previous reports, silica coatedpaper was used for paper spray, rather than chromatography paper orfilter paper, in order to improve the analysis of therapeutic drugs indried blood spots (DBS).

Dichloromethane/isopropanol solvent was identified as an optimal spraysolvent for the analysis. The comparison was made with paper spray usingchromatography paper as substrate with methanol/water as solvent for theanalysis of verapamil, citalopram, amitriptyline, lidocaine andsunitinib in dried blood spots. It was demonstrated that the efficiencyof recovery of the analytes was notably improved with the silica coatedpaper and the limit of quantitation (LOQ) for the drug analysis was 0.1ng mL⁻¹ using a commercial triple quadrupole mass spectrometer. The useof silica paper substrate also resulted in a sensitivity improvement of5-50 fold in comparison with chromatography papers, including theWhatmann ET31 paper used for blood card. Analysis using a handheldminiature mass spectrometer Mini 11 gave LOQs of 10-20 ng mL-1 for thetested drugs, which is sufficient to cover the therapeutic ranges ofthese drugs.

a. Introduction

Accurate measurement of therapeutic drugs and their metabolites in bloodplays an important role in drug discovery and disease therapy. Storingwhole blood samples as the dried blood spots (DBS) on paper is beingadopted for the analysis of drugs in blood. In comparison withconventional way of collecting blood with test tubes, DBS has somespecial advantages including small sample volume (typically less than 50μL), improved chemical stability for many analytes in blood and easysample storage and transfer at ambient temperatures (Rao, R. N.; Maurya,P. K.; Ramesh, M.; Srinivas, R.; Agwane, S. B. Biomed. Chromat. 2010,24, 1356-1364; and Li, W. K.; Zhang, J.; Tse, F. L. S. Biomed. Chromat.2011, 25, 258-277). DBS analysis can be used for a wide range ofapplications, including toxicology (Barfield, M.; Spooner, N.; Lad, R.;Parry, S.; Fowles, S. J. Chromatogr. B: Analyt. Technol. Biomed. LifeSci. 2008, 870, 32-37) and pharmacokinetics studies (Lawson, G.; Tanna,S.; Mulla, H.; Pandya, H. J. Pharm. Pharmacol. 2009, 61, A33; Suyagh, M.F.; Laxman, K. P.; Millership, J.; Collier, P.; Halliday, H.; McElnay,J. C. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 2010, 878,769-776; and Spooner, N.; Lad, R.; Barfield, M. Anal. Chem. 2009, 81,1557-1563) for drug discovery as well as therapeutic drug monitoring(TDM) to assist in dosage optimization during therapy (Ntale, M.;Mahindi, M.; Ogwal-Okeng, J. W.; Gustafsson, L. L.; Beck, O. J.Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 859, 137-140;Lejeune, D.; Souletie, I.; Houze, S.; Le Bricon, T.; Le Bras, J.;Gourmel, B.; Houze, P. J. Pharm. Biomed. Anal. 2007, 43, 1106-1115;Ronn, A. M.; Lemnge, M. M.; Angelo, H. R.; Bygbjerg, I. C. Therap. DrugMonitor. 1995, 17, 79-83; Tawa, R.; Hirose, S.; Fujimoto, T. J.Chromatogr. B: Biomed. Appl. 1989, 490, 125-132; Croes, K.; McCarthy, P.T.; Flanagan, R. J. J. Anal. Toxicol. 1994, 18, 255-260; Heine, R.;Rosing, H.; van Gorp, E. C. M.; Mulder, J. W.; van der Steeg, W. A.;Beijnen, J. H.; Huitema, A. D. R. J. Chromatogr. B: Analyt. Technol.Biomed. Life Sci. 2008, 867, 205-212; Koal, T.; Burhenne, H.; Romling,R.; Svoboda, M.; Resch, K.; Kaever, V. Rapid Commun. Mass Spectrom.2005, 19, 2995-3001; Cheung, C. Y.; van der Heijden, J.; Hoogtanders,K.; Christiaans, M.; Liu, Y. L.; Chan, Y. H.; Choi, K. S.; van de Plas,A.; Shek, C. C.; Chau, K. F.; Li, C. S.; van Hooff, J.; Stolk, L.Transplant Int. 2008, 21, 140-145; Coombes, E. J.; Gamlen, T. R.;Batstone, G. F.; Leigh, P. N. Ann. Clin. Biochem. 1984, 21, 519-522;Fujimoto, T.; Tsuda, Y.; Tawa, R.; Hirose, S. Clin. Chem. 1989, 35,867-869; and Li, P. K.; Lee, J. T.; Conboy, K. A.; Ellis, E. F. Clin.Chem. 1986, 32, 552-555)

Regardless of how blood samples are collected or stored, similarprocedures have been applied for the chemical analysis of thetherapeutic drugs in blood. The analytes are extracted from the bloodsample using organic solvents, then separated using chromatography andanalyzed using mass spectrometry (MS), ultraviolet (UV), fluorescence(FL), or immunoassay. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) has been the mainstream method for the quantitation of drugsin blood (Taylor, P. J.; Tai, C.-H.; Franklin, M. E.; Pillans, P. I.Clin. Biochem. 2011, 44, 14-20; Saint-Marcoux, F.; Sauvage, F.-L.;Marquet, P. Anal. Bioanal. Chem. 2007, 388, 1327-1349; Korecka, M.;Shaw, L. M. Ann. Transplant. 2009, 14, 61-72; and Checa, A.; Oliver, R.;Hernandez-Cassou, S.; Saurina, J. Anal. Chim. Acta 2009, 647, 1-13).High sensitivity and selectivity are obtained with LC-MS/MS forquantitative analysis of therapeutic drugs in blood. The standardprocedure involves complex sample preparation and analyte separationprior to the MS analysis. These steps are essential for minimizingmatrix effects and improving the detection limits (Breadmore, M. C.;Theurillat, R.; Thormann, W. Electrophoresis 2004, 25, 1615-1622; andLee, E. D.; Henion, J. D. Rapid Commun. Mass Spectrom. 1992, 6,727-733), but could take about 30 minutes to several hours. This timeframe might not be rate determining for in-lab analysis of many sampleswhere parallel high throughput approaches can be used, but could be sowhen small numbers of samples are analyzed and rapid decision making isrequired. Used in this way, LC-MS/MS can be applied to a wide range ofdrug compounds; however, this method must be performed in analyticallaboratories and only by experienced chemists.

For analysis of therapeutic drugs in DBS using paper spray, the drugcompounds are extracted from the blood matrix by the wetting solution.The extent of the extraction, relative to other compounds eluting fromthe DBS, has a direct impact on the sensitivity of the analysis. Theaffinity of the drug compounds to a paper substrate also affects theextraction efficiency and causes loss of compound during transport tothe paper tip by the spray solvent and inefficiency in ionization. Thechemical properties of the solvent need to be optimized for theextraction and transfer process, but they also need to be suitable forthe spray ionization process, which is important to the MS analysissensitivity. The selection of the combination of a paper substrate and asolvent is important to the overall performance of the paper spray MSanalysis.

Generally, chromatography paper has mainly been used for paper spraysubstrates with methanol/water as the wetting and spray solvent. ThisExample investigates the use of silica-coated paper for the analysis oftherapeutic drugs in dried blood spots. Silica is by far the most widelyused matrix for chromatographic separation owing to its chemical andmechanical stability, variable pore size, well documented chemistry forsurface modification, and excellent demonstrated performance forseparations. Silica-coated paper is an excellent substrate for resolvinga wide variety of compounds (Marinetti, G. V. In Lipid ChromatographicAnalysis; Wuthier, R. E., Ed.; Marcel Dekker: New York, 1976; Vol. 1, pp59-109; Valadon, L. R. G.; Mummery, R. S. Phytochemistry 1972, 11,413-414; and Egan, R. W. Anal. Biochem. 1975, 68, 654-657).

In this Example, paper coated with silica gel was adopted as the paperspray substrate and relatively less polar solvents were used. Theselection of the substrate/solvent systems was first optimized for druganalysis using a commercial triple quadrupole mass spectrometer and theperformance using a handheld ion trap mass spectrometer (Mini 11; Gao,L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang, Z. Anal. Chem.2008, 80, 7198-7205; Hou, K. Y.; Xu, W.; Xu, J. A.; Cooks, R. G.;Ouyang, Z. Anal. Chem. 2011, 83, 1857-1861; and Sokol, E.; Noll, R. J.;Cooks, R. G.; Beegle, L. W.; Kim, H. I.; Kanik, I. Int. J. MassSpectrom. 2011, In Press, Corrected Proof) was then characterized for aset of therapeutic drugs from DBSs. The combination of the paper spraywith a miniature mass spectrometer is of a great interest forpoint-of-care applications where small-size equipment and simpleoperational procedures are highly desirable. In the Mini 11 handheldmass spectrometer, a single stage discontinuous atmospheric pressureinterface (DAPI; Gao, L.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2008, 80,4026-4032) was used with an ion trap. A TSQ (Thermo Scientific, Inc.,San Jose, Calif.) with multiple differential pumping stages and triplequadrupole mass analyzers can provide 50 times better LOQ for theanalysis of therapeutic drugs in blood using paper spray.

b. Materials

The procedure of paper spray used in this Example was similar to thatpreviously reported (Wang, H.; Liu, J. J.; Cooks, R. G.; Ouyang, Z.Angew. Chem., Int. Ed. 2010, 49, 877-880; Wang, H.; Manicke, N. E.;Yang, Q.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2011,83, 1197-1201; Manicke, N. E.; Yang, Q. A.; Wang, H.; Oradu, S.; Ouyang,Z.; Cooks, R. G. Int. J. Mass Spectrom. 2011, 300, 123-129; and Liu, J.;Wang, H.; Manicke, N. E.; Lin, J.-M.; Cooks, R. G.; Ouyang, Z. Anal.Chem. 2010, 82, 2463-2471). Briefly, the standards used herein,including verapamil, sunitinib, citalopram, amitriptyline, andlidocaine, were prepared as follows: drug solutions at 100×concentration were prepared by dilution of the stock solutions into 1:1methanol/water. The 100× standards were then spiked into bovine wholeblood (with sodium citrate as anticoagulant, Innovative Research, Novi,Mich.) by pipetting 5 μL of the standard into 495 μL of blood. The bloodsamples with lower concentrations of drugs were prepared serially bydiluting 40 μL of higher drug content blood samples with 360 μL ofblood. The concentrations of the drugs in the final blood samples were0.01, 0.1, 1, 10, 1000, and 10,000 ng mL⁻¹, respectively. DBS sampleswere prepared by spotting a fixed volume (5 μL) of blood onto the papersubstrate and drying for at least 4 h at room temperature. Samples werestored at room temperature in a sealed plastic bag containing desiccant.

For paper spray, the DBS substrate was cut into a triangle (10 mm heightand 5 mm base width). A copper clip was used to hold the paper triangleand to apply the high voltage needed for the spray. The distance betweenthe tip of the paper triangle and the inlet to the mass spectrometer wasabout 5 mm. The silica-coated ion-exchange paper Grade SG81 (0.27 mmthick) and the chromatography paper Grade 4 (0.21 mm thick) and GradeET31 (0.50 mm thick) were purchased from Whatman International Ltd.(Maidstone, England) and used without further chemical treatment. TheGrade 4 chromatography paper is of similar thickness to thesilica-coated paper while the thicker Grade ET31 is used for making thecommercial blood cards (Whatman FTA DMPK-C card). The 9:1 methanol/water(v/v) solvent has been found to be generally optimal for analysis ofdrugs in DBS on the cellulose filter papers. The MS analysis was carriedout in positive ion mode with a spray voltage at 3.5 kV. The LOQ valuefor each drug was defined as the lowest concentration within the set oflinear responses. Three replicate measurements were made for eachsample.

Images of surfaces of the chromatography paper (Whatman Grade 4, 0.21 mmthick) and the silica-coated paper (Whatman Grade SG81, 0.27 mm thick))were recorded using a FEI NOVA nanoSEM field emission scanning electronmicroscope (SEM, FEI Company, Hillsboro, Oreg.). The substrates weresputter-coated with platinum for 1.0 min before the analysis and theaccelerating voltage for the Everhart-Thornley detector (ETD, routineimaging) or through-the-lens detector (TLD, highmagnification/resolution imaging) was 5 kV with a working distance ofabout 5.0 mm.

A TSQ Quantum Access Max (Thermo Scientific, San Jose, Calif.), operatedin the selected reaction monitoring (SRM) mode was used and specificproduct ions produced by collision-induced dissociation (CID) weremonitored. The XCALIBUR software (MS control software) was used forcontrol of the TSQ Quantum Access Max MS system and data acquisition.Argon gas (99.995% purity) was used as collision gas. The temperature MSinlet capillary was 300° C. The SRM and instrumental parameters used forthe drug compounds were as follows: verapamil: m/z 455→303, tube lens 97V, Q2 offset (collision energy) 28 V; sunitinib: m/z 399→283, tube lens116 V, Q2 offset 28 V; citalopram: m/z 325→109, tube lens 121 V, Q2offset 28 V; amitriptyline: m/z 278→233, tube lens 108 V, Q2 offset 17V; lidocaine: m/z 235→86, tube lens 94 V, Q2 offset 18 V.

The Mini 11 (Gao, L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang,Z. Anal. Chem. 2008, 80, 7198-7205; Hou, K. Y.; Xu, W.; Xu, J. A.;Cooks, R. G.; Ouyang, Z. Anal. Chem. 2011, 83, 1857-1861; and Sokol, E.;Noll, R. J.; Cooks, R. G.; Beegle, L. W.; Kim, H. I.; Kanik, I. Int. J.Mass Spectrom. 2011, In Press, Corrected Proof) a handheld rectilinearion trap mass spectrometer, has a discontinuous atmospheric pressureinterface (DAPI; Gao, L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.;Ouyang, Z. Anal. Chem. 2008, 80, 7198-7205; and Hou, K. Y.; Xu, W.; Xu,J. A.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2011, 83, 1857-1861) and apumping system with a 10 L/s trubomolecular pump (Pfeiffer HiPace 10,Pfeiffer Vacuum Inc., Nashua, N.H.) and a 5 L/min diaphragm pump(1091-N84.0-8.99, KNF Neuberger Inc., Trenton, N.J.). The flowrestricting capillary in the DAPI is of 5 cm length and 250 μm I.D. TheDAPI was opened for 12 ms to introduce the ions generated by paper sprayand the mass analysis was performed 600 ms after the DAPI was closed andthe vacuum had reached an approximate level. The precursor ions weretrapped at an RF voltage of 275 V_(p-p) at 1.0 MHz and the product ionswere analyzed with a RF scan from 206 V_(p-p) to 4,500 V_(p-p) and aresonance ejection at q=0.70. The frequency and the amplitude of theexcitation signal for the CID were as follows: amitriptyline, 99.40 kHzand 0.39 V_(p-p); citalopram: 84.60 kHz and 0.63 V_(p-p); lidocaine:78.76 kHz and 1.22 V_(p-p); sunitinib: 84.89 kHz and 0.73 V_(p-p);verapamil: 80.58 kHz and 0.37 V_(p-p).

c. Results

SEM images of the substrate surfaces are shown in FIG. 55. Thechromatography paper (FIG. 55A) was shown to have a framework ofcellulosic fibers, each of a diameter 10-20 μm. As previously reportedby Roberts (The Chemistry of Paper; The Royal Society of Chemistry:Letchworth, U K, 1996) strong connections at the points of contactbetween two cellulose fibers were observed, which are due to thehydrogen bonding between the polysaccharides at the fiber surfaces. Thesilica-coated paper (FIG. 55B) has a similar cellulosic framework butthe pores are filled with silica gel particles of diameters 1-5 μm (FIG.55C). Bovine blood of 5.0 μL was deposited onto the paper substrates toform dried blood spots (ca. 7.0 mm diameter). The cellulosic frameworkin the chromatography paper could still be seen except that some smallpores were blocked by the dried blood (FIG. 55D); however, the surfaceof the silica coated paper was completely covered by the dried blood(FIG. 55E). Diffusion of the blood through the paper substrates was alsoexamined as shown in FIGS. 55F-55I. The colors of the top and bottomsides of the paper substrate within the DBS area are similar for thechromatography paper (FIGS. 55F and 55G), but significantly differentfor the silica-coated paper (FIGS. 55H and 55I). The color of the topside of the silica-coated paper substrate is much darker than that ofthe bottom side, which is due to a poor diffusion of the blood throughthe substrate with the framework pores blocked by the silica gelparticles. A relatively large percentage of the blood sample stayed onthe top side of the silica-coated paper substrate, which helped toimprove the efficiency of analyte elution during the paper sprayprocess. Though the blood distribution was less homogenous on thissubstrate, no unexpected sample degradation was observed after they hadbeen stored in a sealed plastic bag for days.

As discussed herein, the paper spray process involves analyte elution aswell as spray ionization. The solvent properties affect not only theextraction of the analytes from the dried blood spots, but also theirtransfer across the paper as well as the ion formation during the spray.For the silica-coated paper, pure solvents with a range of polaritieswere first investigated, including water, hexane, dichloromethane,methanol, ethanol, isopropanol, butyl alcohol. FIG. 56A comparesefficiency of sampling and ionization of verapamil ((M+H)⁺, m/z 455) indried blood spots (500 ng mL⁻¹) using the silica-coated paper substrate.The intensity of the fragment ion m/z 303 was monitored using the triplequadrupole operated in SRM mode. With a spray voltage of 3.5 kV, thelowest signal intensities were observed with water and hexane, whichhave the highest and lowest solvent polarities, respectively. Signalintensities two orders of magnitude greater were observed withdichloromethane, while the best intensities were obtained with thealcohols, viz. methanol, ethanol, isopropanol, and butyl alcohol. Amongthese solvents, isopropanol was the best as a pure solvent for theefficient extraction and spray ionization of verapamil.

It is well-known that the electrospray process is highly dependent onthe polarity and the volatility of the solvent. Typically solventmixtures are used to optimize the spray process for analysis of targetanalytes (Tian, Z. X.; Kass, S. R. J. Am. Chem. Soc. 2008, 130,10842-1084; Lavarone, A. T.; Jurchen, J. C.; Williams, E. R. J. Am. Soc.Mass Spectrom. 2000, 11, 976-985; Venter, A. R.; Kamali, A.; Jain, S.;Bairu, S. Anal. Chem. 2010, 82, 1674-1679; and Li, J. W.; Dewald, H. D.;Chen, H. Anal. Chem. 2009, 81, 9716-9722). Addition of a non- orless-polar solvent component of low boiling point helps to enhance thegeneration of droplets of suitable size during the spray and facilitatestheir subsequent desolvation (Kebarle, P.; Tang, L. Anal. Chem. 1993,65, A972-A986). Paper spray is expected to share similar spraycharacteristics with electrospray but the efficiency of the analyteextraction step needs also to be considered in the selection of solventcomposition. After finding isopropanol as the best pure solvent forpaper spray with silica-coated substrates, dichloromethane was mixedwith isopropanol to produce solvents with increased volatility. Thedirect sampling ionization of the verapamil in the dried blood spots wasperformed using a set of isopropanol/dichloromethane solvents indifferent ratios, as shown in FIG. 56B. The best signal intensity wasobserved with 10% (v/v) of isopropanol in the solvent mixture for thesilica-coated paper. Mixtures of dichloromethane with methanol, ethanol,and butyl alcohol have also been tested and similar trends wereobserved. Among these solvents, 9:1 dichloromethane/isopropanol (v/v)gave the optimal performance with silica-coated paper for verapamil,citalopram, amitriptyline, lidocaine and sunitinib. These solvents werealso tested with chromatography paper substrates (Grade 4 and ET31papers) but the signal intensity was one order of magnitude lower thanthat with 9:1 methanol/water solvent in this case.

The elution efficiency was also characterized for the threesolvent/substrate systems, 9:1 dichloromethane/isopropanol (v/v) forsilica-coated paper and 9:1 methanol/water (v/v) for Grade 4 and GradeET31 chromatography papers, using both pure verapamil sample spots anddried blood spots containing verapamil. The pure verapamil sample spotswere prepared by dropping 5 μL water solution containing verapamil (500ng mL⁻¹) onto the paper and drying the substrate completely. The voltagewas applied and solvent added multiple times to produce many paper sprayevents using the same substrate bearing a single sample spot while thesignal of fragment ion m/z 303 was monitored. Spray solvent of 25 μL wasused each time and the solvent was not added until the monitored ionsignal decreased to a minimum, when the spray solvent was alsoexhausted. Solvent consumption took 5-8 s fordichloromethane/isopropanol on silica-coated paper but 40-70 s formethanol/water on chromatography paper substrates. For both pureanalytes and blood samples, much higher peak intensities were wasobserved for the silica-coated paper substrate (FIGS. 57A and 57D). Most(˜70%) of the verapamil, was eluted during the first elution step of thepaper spray process. A relatively even elution pattern was observed forthe Grade 4 chromatography paper with low peak intensities for verapamil(FIGS. 57B and 57E), although an increasing and then decreasing trendexists. Interestingly, a significant increase in analyte in the 2^(nd)(FIG. 57C) and 5^(th) (FIG. 57F) elution was observed with the GradeET31 paper for the pure and the blood samples, respectively. Presumably,this is due to the relatively larger substrate volume, which requiressufficient wetting of the substrate and transfer of the analytes to thesubstrate tip for the paper spray.

Similar procedure was applied to dried blood spots containing verapamil.The blood spots were each prepared with 5 μL of blood sample containing500 ng mL⁻¹ verapamil. The matrix in the dried blood spots is much morecomplex than that in the pure verapamil sample spots. For silica-coatedpaper with 9:1 dichloromethane/isopropanol, analyte release during thefirst paper spray event was still dominant, although the peak intensityfor verapamil decreased about 3 times, presumably due to the matrixeffect. For chromatography papers with methanol/water, no significantdifference in elution pattern was observed except that the maxima of theverapamil detected appeared at later elutions (FIGS. 57D and 57F). Therewas also about an order of magnitude difference in the best signalintensities observed for these three systems in favor of the hydrophobicsubstrate.

Without be limited by any particular theory or mechanism of action,several factors could account for the difference between these twosubstrate/solvent systems and the improvement with the silica-coatedpaper substrate. The blocking of the pores in the cellulosic frameworkby the silica resulted in a more concentrated sample on the top surfaceof the substrate and also a less binding interaction between the analyteand the cellulose. This helps to improve analyte elution during thepaper spray process. The polarity of the solvent systems might also playan important role. The polarity of methanol/water solvent (9:1, v/v) ishigher than that of dichloromethane/isopropanol solvent (9:1, v/v)(polarity index: water 10.2, methanol 5.1, isopropanol 3.9 anddichloromethane 3.1). Low polarity organic compounds, such as verapamil,dissolve better in dichloromethane/isopropanol solvent, which makes theextraction of these chemicals from dried blood more efficient.

The performance of the improved substrate/solvent system wascharacterized for quantitative analysis of therapeutic drugs in wholeblood samples using paper spray. Dried blood spots on paper spraysubstrates were prepared by depositing 5 μL whole blood containingverapamil at a concentration from 0.01 ng mL⁻¹ to 10,000 ng mL⁻¹ anddrying the sample on substrate completely. MS analysis was performedusing the triple quadrupole mass spectrometer and the peak intensity ofm/z 303 from verapamil ((M+H)⁺, m/z 455) was recorded. As shown in FIG.58A, an LOQ of 0.1 ng mL⁻¹ was observed for silica-coated substrate with9:1 (v/v) dichloromethane/isopropanol, which is about two orders ofmagnitude better than that for Grade 4 (FIG. 58B) and Grade ET31 (FIG.58C) chromatography papers with 9:1 (v/v) methanol/water. Othertherapeutic drugs in whole blood, including sunitinib, citalopram,amitriptyline, and lidocaine, were also analyzed and an LOQ of 0.1 ngmL⁻¹ was obtained for each of them using paper spray with silica-coatedpaper.

In this Example, paper spray using the silica paper substrates was alsocharacterized using a miniature rectilinear ion trap mass spectrometer,Mini 11. This was done by quantitative analysis of verapamil, sunitinib,citalopram, amitriptyline, and lidocaine in dried blood spots. MS/MSanalysis was performed using the procedure previously described (Gao,L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang, Z. Anal. Chem.2008, 80, 7198-7205; Hou, K. Y.; Xu, W.; Xu, J. A.; Cooks, R. G.;Ouyang, Z. Anal. Chem. 2011, 83, 1857-1861; and Sokol, E.; Noll, R. J.;Cooks, R. G.; Beegle, L. W.; Kim, H. I.; Kanik, I. Int. J. MassSpectrom. 2011, In Press, Corrected Proof) to acquire the intensities ofthe characteristic fragment ions from the drug compounds.

FIGS. 59A-59B shows the low concentration regions (1 ng mL⁻¹ to 1,000 ngmL⁻¹) of the linear ranges for quantitation of lidocaine (fragment ionm/z 86) and verapamil (fragment ion m/z 165). LOQ values for lidocaineand verapamil are 20 ng mL⁻¹ and 10 ng mL⁻¹, respectively, approximatelytwo orders of magnitude higher than achieved using the benchtop triplequadruple. The spectra recorded at LOQs for these two drugs are shown inFIGS. 59C-59D. Good linearity was also obtained in the ranges from theirLOQs to 1 μg/mL.

A comparison study was done for silica-coated paper and chromatographypaper (Whatman Grade ET31, 0.50 mm thick) using paper spray and the Mini11 for analysis of therapeutic drugs in dried blood spots. The LOQsobtained and the therapeutic windows for lidocaine, amitriptyline,sunitinib, verapamil, and citalopram are listed in Table 6.

TABLE 6 Comparison of LOQ values^(a) for some typical drugs between ET31 chromatography paper and silica coated paper with Mini 11. LOQ withChromatography LOQ with Therapeutic Precursor Product paper silicacoated range Drug ion ion (ng mL⁻¹) paper (ng mL⁻¹) (ng mL⁻¹) lidocaine235 86 100 20 1,000-6,000  amitriptyline 278 233 100 10 50-200 sunitinib399 326 500 10 20-200 verapamil 455 165 100 10 50-250 citalopram 325 109100 10 10-200 ^(a)LOQ for each drug is defined as the lowestconcentration within the set of linear responses in the sensitivity plotas shown in FIGs. 58 and 59A-59B. Three replicates for each sample.

Using the chromatography paper with 9:1 methanol/water, the performanceof the paper spray/Mini 11 system was only good enough to cover thetherapeutic range for lidocaine, but not for the other 5 drugs in DBSs.With the silica paper substrate and 9:1 dicholoramethane/isopropanol,improvements of 5-50 fold were obtained in LOQs for these drugs, whichmade the performance of the miniature systems adequate for theirquantitative analysis from dried blood spots.

d. Conclusions

Paper spray with a silica coated paper substrate was characterized usinga commercial triple quadrupole and a home-built miniature ion trap massspectrometer for analysis of therapeutic drugs in dried blood spots. Theoverall analysis efficiency can be greatly improved by using low boilingand low polarity solvent, 9:1 dichloromethane/isopropanol (v/v). The LOQof analysis of a set of therapeutic drugs in dried blood spots,including verapamil, citalopram, amitriptyline, lidocaine, andsunitinib, was obtained as low as 0.1 ng mL⁻¹ with the commercial triplequadrupole and 10˜20 ng mL⁻¹ with a miniature ion trap massspectrometer. The triple quadrupole is inherently better suited to SRManalysis than is an ion trap instrument so this factor as well as thereduced performance of the Mini MS is responsible for the difference.Compared to chromatography paper, in each case there is a 5-50 foldimprovement with the silica paper substrate.

1-90. (canceled)
 91. A sample analysis system comprising: a cartridgecomprising a porous substrate that tapers to a tip and an electrodeconnected to the porous substrate; and a mass spectrometer.
 92. Thesystem according to claim 91, wherein the cartridge is configured suchthat the tip of the porous substrate does not extend beyond an end ofthe cartridge.
 93. The system according to claim 91, wherein thecartridge further comprises a bottom portion and a detachable cover, andthe cartridge is configured such that the bottom portion is configuredto hold the porous substrate.
 94. The system according to claim 93,wherein the cartridge is configured such that voltage is not supplied tothe porous material in the cartridge until the detachable cover isconnected to the bottom portion.
 95. The system according to claim 93,wherein the cartridge further comprises a solvent port.
 96. The systemaccording to claim 95, wherein the solvent port is configured forcontinuous supply of solvent to the porous substrate.
 97. The systemaccording to claim 95, wherein the solvent port is configured for supplyof only a discrete amount of solvent to the porous substrate such thatporous substrate is discrete from a flow of solvent.
 98. The systemaccording to claim 91, wherein the system is configured such that thecartridge is positioned with respect to an inlet of the massspectrometer so that the tip of the porous substrate is aligned with theinlet of the mass spectrometer.
 99. The system according to claim 91,wherein the porous substrate is paper.
 100. The system according toclaim 99, wherein the paper is filter paper.
 101. A method for analyzinga sample, the method comprising: applying a sample to a porous substratethat tapers to a tip; loading the porous substrate into a cartridgecomprising an electrode such that the electrode is connected to theporous substrate; applying voltage to the porous substrate to generatesample ions; and analyzing the sample ions.
 102. The method according toclaim 101, wherein the cartridge is configured such that the tip of theporous substrate does not extend beyond an end of the cartridge. 103.The method according to claim 101, wherein the cartridge furthercomprises a bottom portion and a detachable cover, and the cartridge isconfigured such that the bottom portion is configured to hold the poroussubstrate.
 104. The method according to claim 103, wherein voltage isnot supplied to the porous material in the cartridge until thedetachable cover is connected to the bottom portion.
 105. The methodaccording to claim 103, wherein the cartridge further comprises asolvent port.
 106. The method according to claim 105, wherein the methodfurther comprises supplying solvent via the solvent port to the poroussubstrate in the cartridge prior to applying the voltage to the poroussubstrate.
 107. The method according to claim 101, wherein the methodfurther comprises supplying solvent to the porous substrate prior to theporous substrate being loading into the cartridge.
 108. The methodaccording to claim 101, wherein analyzing comprising introducing thesample ions into a mass spectrometer and analyzing the sample ions inthe mass spectrometer.
 109. The method according to claim 101, whereinthe porous substrate is paper.
 110. The system according to claim 109,wherein the paper is filter paper.